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Neurologist (brain, nervous system), Psychiatrist, Sleep Medicine Specialist
26 years of experience
Accepting new patients
Video profile

Credentials

Education ?

Medical School Score Rankings
University of Massachusetts (1986)
  •  
Top 25%

Awards & Distinctions ?

Awards  
Patients' Choice Award (2012)
Compassionate Doctor Recognition (2013, 2015)
Top 10 Doctor - City (2014)
Philadelphia, PA
Neurologist
On-Time Doctor Award (2014 - 2015)
Appointments
University of Pennsylvania
Clinical Associate Professor of Neurology and Medicine Medical Director, Penn Sleep Centers
Associations
American Board of Psychiatry and Neurology

Affiliations ?

Dr. Cantor is affiliated with 8 hospitals.

Hospital Affiliations

Score

Rankings

  • Thomas Jefferson University Hospital
    111 S 11th St, Philadelphia, PA 19107
    •  
    Top 25%
  • Hospital of the University of PA
    3400 Spruce St, Philadelphia, PA 19104
    •  
    Top 25%
  • Pennsylvania Hospital University PA Health System
    800 Spruce St, Philadelphia, PA 19107
    •  
    Top 25%
  • University of PA Medical Center/Presbyterian
    51 N 39th St, Philadelphia, PA 19104
    •  
    Top 50%
  • Graduate Hospital
    1800 Lombard St, Philadelphia, PA 19146
    •  
  • Clinical Practices of the University of Pennsylvania
  • Pennsylvania Hospital
  • Presbyterian Medical Center Of The University Of Pennsylvania Health System
  • Publications & Research

    Dr. Cantor has contributed to 232 publications.
    Title Noninvasive Detection of Fetal Trisomy 21 by Sequencing of Dna in Maternal Blood: a Study in a Clinical Setting.
    Date June 2011
    Journal American Journal of Obstetrics and Gynecology
    Excerpt

    We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA.

    Title Intron Retention Facilitates Splice Variant Diversity in Calcium-activated Big Potassium Channel Populations.
    Date January 2011
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.

    Title Tracking, Tuning, and Terminating Microbial Physiology Using Synthetic Riboregulators.
    Date October 2010
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wild-type cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications.

    Title Bacterial Charity Work Leads to Population-wide Resistance.
    Date September 2010
    Journal Nature
    Excerpt

    Bacteria show remarkable adaptability in the face of antibiotic therapeutics. Resistance alleles in drug target-specific sites and general stress responses have been identified in individual end-point isolates. Less is known, however, about the population dynamics during the development of antibiotic-resistant strains. Here we follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the population's less resistant constituents, in part by producing indole, a signalling molecule generated by actively growing, unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative-stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and whole-genome sequencing reveals that this bacterial altruism is made possible by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other, more vulnerable, cells, enhancing the survival capacity of the overall population in stressful environments.

    Title Hypermethylation of Genes for Diagnosis and Risk Stratification of Prostate Cancer.
    Date June 2009
    Journal Cancer Investigation
    Excerpt

    To identify the relevant CpG sites as molecular markers, for the diagnosis and to distinguish the indolent and aggressive prostate tumors, we have determined the methylation status of 8 genes, including FLNC, EFS, ECRG4, RARB2, PITX2, GSTP1, PDLIM4, and KCNMA1 in 32 nonrecurrent, 32 recurrent primary prostate tumors, and 32 benign prostate tissues using EpiTYPER technology. Specific CpG site hypermethylation of RARB2 and GSTP1 CpG sites were useful for diagnosis of prostate cancer. Furthermore, CpG site hypermethylation of genes FLNC, EFS, ECRG4, PITX2, PDLIM4, and KCNMA1 were associated with prediction of biochemical, local, and systemic recurrence of prostate cancer.

    Title A Network Biology Approach to Aging in Yeast.
    Date February 2009
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    In this study, a reverse-engineering strategy was used to infer and analyze the structure and function of an aging and glucose repressed gene regulatory network in the budding yeast Saccharomyces cerevisiae. The method uses transcriptional perturbations to model the functional interactions between genes as a system of first-order ordinary differential equations. The resulting network model correctly identified the known interactions of key regulators in a 10-gene network from the Snf1 signaling pathway, which is required for expression of glucose-repressed genes upon calorie restriction. The majority of interactions predicted by the network model were confirmed using promoter-reporter gene fusions in gene-deletion mutants and chromatin immunoprecipitation experiments, revealing a more complex network architecture than previously appreciated. The reverse-engineered network model also predicted an unexpected role for transcriptional regulation of the SNF1 gene by hexose kinase enzyme/transcriptional repressor Hxk2, Mediator subunit Med8, and transcriptional repressor Mig1. These interactions were validated experimentally and used to design new experiments demonstrating Snf1 and its transcriptional regulators Hxk2 and Mig1 as modulators of chronological lifespan. This work demonstrates the value of using network inference methods to identify and characterize the regulators of complex phenotypes, such as aging.

    Title Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidy by Massively Parallel Genomic Sequencing of Dna in Maternal Plasma.
    Date January 2009
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

    Title Noninvasive Prenatal Diagnosis of Monogenic Diseases by Digital Size Selection and Relative Mutation Dosage on Dna in Maternal Plasma.
    Date January 2009
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

    Title Digital Pcr for the Molecular Detection of Fetal Chromosomal Aneuploidy.
    Date September 2007
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.

    Title A Tunable Genetic Switch Based on Rnai and Repressor Proteins for Regulating Gene Expression in Mammalian Cells.
    Date September 2007
    Journal Cell
    Excerpt

    Here, we introduce an engineered, tunable genetic switch that couples repressor proteins and an RNAi target design to effectively turn any gene off. We used the switch to regulate the expression of EGFP in mouse and human cells and found that it offers >99% repression as well as the ability to tune gene expression. To demonstrate the system's modularity and level of gene silencing, we used the switch to tightly regulate the expression of diphtheria toxin and Cre recombinase, respectively. We also used the switch to tune the expression of a proapoptotic gene and show that a threshold expression level is required to induce apoptosis. This work establishes a system for tight, tunable control of mammalian gene expression that can be used to explore the functional role of various genes as well as to determine whether a phenotype is the result of a threshold response to changes in gene expression.

    Title Automated Comparative Sequence Analysis by Base-specific Cleavage and Mass Spectrometry for Nucleic Acid-based Microbial Typing.
    Date August 2007
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database (http://pubmlst.org/neisseria) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis, was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.

    Title Rna Visualization in Live Bacterial Cells Using Fluorescent Protein Complementation.
    Date June 2007
    Journal Nature Methods
    Excerpt

    We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.

    Title Plasma Placental Rna Allelic Ratio Permits Noninvasive Prenatal Chromosomal Aneuploidy Detection.
    Date April 2007
    Journal Nature Medicine
    Excerpt

    Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

    Title Engineering a Novel, Stable Dimeric Streptavidin with Lower Isoelectric Point.
    Date April 2007
    Journal Journal of Biotechnology
    Excerpt

    We have engineered a soluble, stable two-chain dimeric streptavidin (TCD) in Escherchia coli. Examination of the three-dimensional structure of streptavidin aided by empirical binding free-energy calculations helped us to select mutations at subunit interfaces that dissociate the native tetramer and stabilize the desired dimer. We chose positions W120, L124, V125 and H127 and mutated them to 120D/124D/125D/127D (TCD-1); 120D/124N/125S/127D (TCD-2); and 120D/124D/125S/127D (TCD-3). The H127D mutation creates electrostatic repulsion that disrupts the dimer-dimer interface, but leaves it very hydrophobic. Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility. Among the three candidates, TCD-2 gave the best result: a stable, active dimer with K(d) for biotin of approximately 1x10(-7)M after purification by gel-filtration chromatography. The experimental results confirm the possibility of rational engineering of low-pI dimeric streptavidins. Reduced-size streptavidin mutants with a net negative charge may be more suitable than antibodies or wild-type streptavidin for the targeting step in radioimmunotherapy because they should clear faster from the bloodstream and the kidney.

    Title Comparative Genome Sequencing of Escherichia Coli Allows Observation of Bacterial Evolution on a Laboratory Timescale.
    Date February 2007
    Journal Nature Genetics
    Excerpt

    We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

    Title Phenotypic Consequences of Promoter-mediated Transcriptional Noise.
    Date January 2007
    Journal Molecular Cell
    Excerpt

    A more complete understanding of the causes and effects of cell-cell variability in gene expression is needed to elucidate whether the resulting phenotypes are disadvantageous or confer some adaptive advantage. Here we show that increased variability in gene expression, affected by the sequence of the TATA box, can be beneficial after an acute change in environmental conditions. We rationally introduce mutations within the TATA region of an engineered Saccharomyces cerevisiae GAL1 promoter and measure promoter responses that can be characterized as being either highly variable and rapid or steady and slow. We computationally illustrate how a stable transcription scaffold can result in "bursts" of gene expression, enabling rapid individual cell responses in the transient and increased cell-cell variability at steady state. We experimentally verify computational predictions that the rapid response and increased cell-cell variability enabled by TATA-containing promoters confer a clear benefit in the face of an acute environmental stress.

    Title Cytosine Methylation Profiles As a Molecular Marker in Non-small Cell Lung Cancer.
    Date December 2006
    Journal Cancer Research
    Excerpt

    Aberrant promoter methylation is frequently observed in different types of lung cancer. Epigenetic modifications are believed to occur before the clinical onset of the disease and hence hold a great promise as early detection markers. Extensive analysis of DNA methylation has been impeded by methods that are either too labor intensive to allow large-scale studies or not sufficiently quantitative to measure subtle changes in the degree of methylation. We used a novel quantitative DNA methylation analysis technology to complete a large-scale cytosine methylation profiling study involving 47 gene promoter regions in 96 lung cancer patients. Each individual contributed a lung cancer specimen and corresponding adjacent normal tissue. The study identified six genes with statistically significant differences in methylation between normal and tumor tissue (P < 10(-6)). We explored the quantitative methylation data using an unsupervised hierarchical clustering algorithm. The data analysis revealed that methylation patterns differentiate normal from tumor tissue. For validation of our approach, we divided the samples to train a classifier and test its performance. We were able to distinguish normal from lung cancer tissue with >95% sensitivity and specificity. These results show that quantitative cytosine methylation profiling can be used to identify molecular classification markers in lung cancer.

    Title Genome-wide Snp Association: Identification of Susceptibility Alleles for Osteoarthritis.
    Date August 2006
    Journal Autoimmunity Reviews
    Excerpt

    The successful identification of genes involved in common human disorders is dependent upon availability of informative sample sets, validated marker panels, a high-throughput scoring technology, and a strategy for combining these resources. We have developed a universal platform based on mass spectrometry (MassARRAY) for analyzing nucleic acids with high precision and accuracy. To fuel this technology we have generated more than 100,000 validated assays for single nucleotide polymorphisms (SNPs) covering virtually all known and predicted human genes, and a large DNA sample bank from more than 50,000 consented diseased (case) and healthy (control) individuals. Taking advantage of MassARRAY's capability for quantitative analysis of nucleic acids, allele frequencies are estimated in sample pools containing large numbers of individual DNAs. Comparing frequencies between case and control pools as a first-pass filtering step is a tremendous advantage in throughput and cost over individual genotyping. We have employed this approach in numerous genome-wide association studies to identify genes implicated in common complex diseases, including osteoarthritis (OA). Access to additional patient samples through collaborations allows us to conduct replication studies that validate true disease genes. These discoveries will expand our understanding of genetic disease predisposition, and our capabilities for early diagnosis and improved therapeutic approaches.

    Title The Use of Genetic Snps As New Diagnostic Markers in Preventive Medicine.
    Date June 2006
    Journal Annals of the New York Academy of Sciences
    Excerpt

    Using an automated mass spectrometric genotyping platform, we have completed more than ten whole-genome SNP scans on phenotypically stratified population pools. The pools are usually constructed to represent one ethnicity, one gender, and one phenotype, classified as strictly as possible. From 28,000 to 91,000 different SNPs are used for each study, and the pools typically contain DNA from 300 different individuals. Significant correlations between SNP allele and phenotype are first reproduced in the pools, then replicated on individual DNA samples (deconvolution of the pools), and then where possible replicated in completely independent populations.

    Title Longevity Determinant Genes: What is the Evidence? What's the Importance? Panel Discussion.
    Date June 2006
    Journal Annals of the New York Academy of Sciences
    Title Dna Conformation on Surfaces Measured by Fluorescence Self-interference.
    Date June 2006
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral self-interference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.

    Title Fast Complementation of Split Fluorescent Protein Triggered by Dna Hybridization.
    Date April 2006
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells.

    Title A Bottom-up Approach to Gene Regulation.
    Date March 2006
    Journal Nature
    Excerpt

    The ability to construct synthetic gene networks enables experimental investigations of deliberately simplified systems that can be compared to qualitative and quantitative models. If simple, well-characterized modules can be coupled together into more complex networks with behaviour that can be predicted from that of the individual components, we may begin to build an understanding of cellular regulatory processes from the 'bottom up'. Here we have engineered a promoter to allow simultaneous repression and activation of gene expression in Escherichia coli. We studied its behaviour in synthetic gene networks under increasingly complex conditions: unregulated, repressed, activated, and simultaneously repressed and activated. We develop a stochastic model that quantitatively captures the means and distributions of the expression from the engineered promoter of this modular system, and show that the model can be extended and used to accurately predict the in vivo behaviour of the network when it is expanded to include positive feedback. The model also reveals the counterintuitive prediction that noise in protein expression levels can increase upon arrest of cell growth and division, which we confirm experimentally. This work shows that the properties of regulatory subsystems can be used to predict the behaviour of larger, more complex regulatory networks, and that this bottom-up approach can provide insights into gene regulation.

    Title Quantitative High-throughput Analysis of Dna Methylation Patterns by Base-specific Cleavage and Mass Spectrometry.
    Date December 2005
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.

    Title Real-time Monitoring of Branched Rolling-circle Dna Amplification with Peptide Nucleic Acid Beacon.
    Date September 2005
    Journal Analytical Biochemistry
    Title Engineered Single-chain Dimeric Streptavidins with an Unexpected Strong Preference for Biotin-4-fluorescein.
    Date August 2005
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.

    Title An Avidin-like Domain That Does Not Bind Biotin is Adopted for Oligomerization by the Extracellular Mosaic Protein Fibropellin.
    Date July 2005
    Journal Protein Science : a Publication of the Protein Society
    Excerpt

    The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.

    Title High-throughput Alternative Splicing Quantification by Primer Extension and Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry.
    Date June 2005
    Journal Nucleic Acids Research
    Excerpt

    Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.

    Title Haplotyping in Biomedicine-practical Challenges.
    Date June 2005
    Journal Nature Biotechnology
    Title A Single Nucleotide Polymorphism Based Approach for the Identification and Characterization of Gene Expression Modulation Using Massarray.
    Date May 2005
    Journal Mutation Research
    Excerpt

    Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes. These changes in expression due to allelic variation are often associated with additional genomic or transcript modifications such as DNA methylation or RNA editing. Here, we describe the use of an integrated genetic analysis platform, based on matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to first, discover coding SNPs (cSNPs); second, use these cSNPs to identify and analyze allele-specific expression; and third, from this knowledge to further analyze methylation patterns as a putative cause for the allele-specific expression. An established model involving allele-specific expression profiles of the human tumor protein 73 (TP73) gene is presented as an example to outline and validate data obtained from the MassARRAY platform. The availability of a single integrated platform to assay stable and dynamic variation at the genomic and transcript level greatly simplifies complex functional genomic studies.

    Title Association of the Numa Region on Chromosome 11q13 with Breast Cancer Susceptibility.
    Date April 2005
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The development of breast cancer is a complex process that involves multiple genes at many stages, from initial cell cycle dysregulation to disease progression. To identify genetic variations that influence this process, we conducted a large-scale association study using a collection of German cases and controls and >25,000 SNPs located within 16,000 genes. One of the loci identified was located on chromosome 11q13 [odds ratio (OR)=1.85, P=0.017]. The initial association was subsequently tested in two independent breast cancer collections. In both sample sets, the frequency of the susceptibility allele was increased in the cases (OR=1.6, P=0.01). The susceptibility allele was also associated with an increase in cancer family history (P=0.1). Fine mapping showed that the region of association extends approximately 300 kb and spans several genes, including the gene encoding the nuclear mitotic apparatus protein (NuMA). A nonsynonymous SNP (A794G) in NuMA was identified that showed a stronger association with breast cancer risk than the initial marker SNP (OR=2.8, P=0.005 initial sample; OR=2.1, P=0.002 combined). NuMA is a cell cycle-related protein essential for normal mitosis that is degraded in early apoptosis. NuMA-retinoic acid receptor alpha fusion proteins have been described in acute promyelocytic leukemia. Although the potential functional relevance of the A794G variation requires further biological validation, we conclude that variations in the NuMA gene are likely responsible for the observed increased breast cancer risk.

    Title High-throughput Mutation Detection Underlying Adaptive Evolution of Escherichia Coli-k12.
    Date February 2005
    Journal Genome Research
    Excerpt

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.

    Title Large-scale Association Study Identifies Icam Gene Region As Breast and Prostate Cancer Susceptibility Locus.
    Date February 2005
    Journal Cancer Research
    Excerpt

    We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

    Title Engineered Riboregulators Enable Post-transcriptional Control of Gene Expression.
    Date January 2005
    Journal Nature Biotechnology
    Excerpt

    Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5'-untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.

    Title Gc/at-content Spikes As Genomic Punctuation Marks.
    Date January 2005
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Large-scale analysis of the GC-content distribution at the gene level reveals both common features and basic differences in genomes of different groups of species. Sharp changes in GC content are detected at the transcription boundaries for all species analyzed, including human, mouse, rat, chicken, fruit fly, and worm. However, two substantially distinct groups of GC-content profiles can be recognized: warm-blooded vertebrates including human, mouse, rat, and chicken, and invertebrates including fruit fly and worm. In vertebrates, sharp positive and negative spikes of GC content are observed at the transcription start and stop sites, respectively, and there is also a progressive decrease in GC content from the 5' untranslated region to the 3' untranslated region along the gene. In invertebrates, the positive and negative GC-content spikes at the transcription start and stop sites are preceded by spikes of opposite value, and the highest GC content is found in the coding regions of the genes. Cross-correlation analysis indicates high frequencies of GC-content spikes at transcription start and stop sites. The strong conservation of this genomic feature seen in comparisons of the human/mouse and human/rat orthologs, and the clustering of genes with GC-content spikes on chromosomes imply a biological function. The GC-content spikes at transcription boundaries may reflect a general principle of genomic punctuation. Our analysis also provides means for identifying these GC-content spikes in individual genomic sequences.

    Title Simultaneous Quantitative and Allele-specific Expression Analysis with Real Competitive Pcr.
    Date November 2004
    Journal Bmc Genetics
    Excerpt

    BACKGROUND: For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis. RESULTS: A transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies. CONCLUSIONS: This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.

    Title Large-scale Validation of Single Nucleotide Polymorphisms in Gene Regions.
    Date September 2004
    Journal Genome Research
    Excerpt

    Genome-wide association studies using large numbers of bi-allelic single nucleotide polymorphisms (SNPs) have been proposed as a potentially powerful method for identifying genes involved in common diseases. To assemble a SNP collection appropriate for large-scale association, we designed assays for 226,099 publicly available SNPs located primarily within known and predicted gene regions. Allele frequencies were estimated in a sample of 92 CEPH Caucasians using chip-based MALDI-TOF mass spectrometry with pooled DNA. Of the 204,200 designed assays that were functional, 125,799 SNPs were determined to be polymorphic (minor allele frequency > 0.02), of which 101,729 map uniquely to the human genome. Many of the commonly available RefSNP annotations were predictive of polymorphic status and could be used to improve the selection of SNPs from the public domain for genetic research. The set of uniquely mapping, polymorphic SNPs is located within 10 kb of 66% of known and predicted genes annotated in LocusLink, which could prove useful for large-scale disease association studies.

    Title Ms Analysis of Single-nucleotide Differences in Circulating Nucleic Acids: Application to Noninvasive Prenatal Diagnosis.
    Date August 2004
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian beta-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the beta-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical beta-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.

    Title Mining Disease Susceptibility Genes Through Snp Analyses and Expression Profiling Using Maldi-tof Mass Spectrometry.
    Date August 2004
    Journal Journal of Proteome Research
    Excerpt

    To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis. Here, we provide a brief review of the technology and its applications to disease gene discovery.

    Title Effect of Streptavidins with Varying Biotin Binding Affinities on the Properties of Biotinylated Gramicidin Channels.
    Date August 2004
    Journal Biochemistry
    Excerpt

    The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.

    Title Programmable Cells: Interfacing Natural and Engineered Gene Networks.
    Date July 2004
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Novel cellular behaviors and characteristics can be obtained by coupling engineered gene networks to the cell's natural regulatory circuitry through appropriately designed input and output interfaces. Here, we demonstrate how an engineered genetic circuit can be used to construct cells that respond to biological signals in a predetermined and programmable fashion. We employ a modular design strategy to create Escherichia coli strains where a genetic toggle switch is interfaced with: (i) the SOS signaling pathway responding to DNA damage, and (ii) a transgenic quorum sensing signaling pathway from Vibrio fischeri. The genetic toggle switch endows these strains with binary response dynamics and an epigenetic inheritance that supports a persistent phenotypic alteration in response to transient signals. These features are exploited to engineer cells that form biofilms in response to DNA-damaging agents and cells that activate protein synthesis when the cell population reaches a critical density. Our work represents a step toward the development of "plug-and-play" genetic circuitry that can be used to create cells with programmable behaviors.

    Title Quantitative Analysis of Nucleic Acids--the Last Few Years of Progress.
    Date July 2004
    Journal Journal of Biochemistry and Molecular Biology
    Excerpt

    DNA and RNA quantifications are widely used in biological and biomedical research. In the last ten years, many technologies have been developed to enable automated and high-throughput analyses. In this review, we first give a brief overview of how DNA and RNA quantifications are carried out. Then, five technologies (microarrays, SAGE, differential display, real time PCR and real competitive PCR) are introduced, with an emphasis on how these technologies can be applied and what their limitations are. The technologies are also evaluated in terms of a few key aspects of nucleic acids quantification such as accuracy, sensitivity, specificity, cost and throughput.

    Title Whole-genome Annotation by Using Evidence Integration in Functional-linkage Networks.
    Date June 2004
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The advent of high-throughput biology has catalyzed a remarkable improvement in our ability to identify new genes. A large fraction of newly discovered genes have an unknown functional role, particularly when they are specific to a particular lineage or organism. These genes, currently labeled "hypothetical," might support important biological cell functions and could potentially serve as targets for medical, diagnostic, or pharmacogenomic studies. An important challenge to the scientific community is to associate these newly predicted genes with a biological function that can be validated by experimental screens. In the absence of sequence or structural homology to known genes, we must rely on advanced biotechnological methods, such as DNA chips and protein-protein interaction screens as well as computational techniques to assign putative functions to these genes. In this article, we propose an effective methodology for combining biological evidence obtained in several high-throughput experimental screens and integrating this evidence in a way that provides consistent functional assignments to hypothetical genes. We use the visualization method of propagation diagrams to illustrate the flow of functional evidence that supports the functional assignments produced by the algorithm. Our results contain a number of predictions and furnish strong evidence that integration of functional information is indeed a promising direction for improving the accuracy and robustness of functional genomics.

    Title A Sense of Life: Computational and Experimental Investigations with Models of Biochemical and Evolutionary Processes.
    Date May 2004
    Journal Omics : a Journal of Integrative Biology
    Excerpt

    We collaborate in a research program aimed at creating a rigorous framework, experimental infrastructure, and computational environment for understanding, experimenting with, manipulating, and modifying a diverse set of fundamental biological processes at multiple scales and spatio-temporal modes. The novelty of our research is based on an approach that (i) requires coevolution of experimental science and theoretical techniques and (ii) exploits a certain universality in biology guided by a parsimonious model of evolutionary mechanisms operating at the genomic level and manifesting at the proteomic, transcriptomic, phylogenic, and other higher levels. Our current program in "systems biology" endeavors to marry large-scale biological experiments with the tools to ponder and reason about large, complex, and subtle natural systems. To achieve this ambitious goal, ideas and concepts are combined from many different fields: biological experimentation, applied mathematical modeling, computational reasoning schemes, and large-scale numerical and symbolic simulations. From a biological viewpoint, the basic issues are many: (i) understanding common and shared structural motifs among biological processes; (ii) modeling biological noise due to interactions among a small number of key molecules or loss of synchrony; (iii) explaining the robustness of these systems in spite of such noise; and (iv) cataloging multistatic behavior and adaptation exhibited by many biological processes.

    Title Analysis of Sequence Variations in Several Human Genes Using Phosphoramidite Bond Dna Fragmentation and Chip-based Maldi-tof.
    Date February 2004
    Journal Genome Research
    Excerpt

    The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.

    Title Native Disulfide Bonds in Plasma Retinol-binding Protein Are Not Essential for All-trans-retinol-binding Activity.
    Date February 2004
    Journal Journal of Proteome Research
    Excerpt

    A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.

    Title Dopamine Agonists and Sleep in Parkinson's Disease.
    Date September 2003
    Journal Neurology
    Excerpt

    Dopaminergic therapy is increasingly recognized as a cause of excessive daytime sleepiness in patients with PD. This adverse effect may be a dose-related phenomenon that is somewhat more likely to occur with dopamine agonists than with levodopa, although all dopaminergic drugs can be sedating. However, medication effect is only one of several causes of somnolence in PD. Other factors include age-related changes in sleep quality, nocturnal motor disturbances, primary sleep disorders such as sleep apnea, medication-induced sleep disruption, and concurrent medical illnesses. There is also increasing evidence that the disease process itself may affect the control of the sleep-wake cycle. Although we have characterized the sleep disturbances in PD, further investigation is needed to define their prevalence and etiology, particularly with respect to the role of dopamine and dopaminergic agents. Clinicians should be alert to the complaint of excessive sleepiness in their patients and should attempt to identify and treat the underlying causes.

    Title Human-mouse Gene Identification by Comparative Evidence Integration and Evolutionary Analysis.
    Date August 2003
    Journal Genome Research
    Excerpt

    The identification of genes in the human genome remains a challenge, as the actual predictions appear to disagree tremendously and vary dramatically on the basis of the specific gene-finding methodology used. Because the pattern of conservation in coding regions is expected to be different from intronic or intergenic regions, a comparative computational analysis can lead, in principle, to an improved computational identification of genes in the human genome by using a reference, such as mouse genome. However, this comparative methodology critically depends on three important factors: (1) the selection of the most appropriate reference genome. In particular, it is not clear whether the mouse is at the correct evolutionary distance from the human to provide sufficiently distinctive conservation levels in different genomic regions, (2) the selection of comparative features that provide the most benefit to gene recognition, and (3) the selection of evidence integration architecture that effectively interprets the comparative features. We address the first question by a novel evolutionary analysis that allows us to explicitly correlate the performance of the gene recognition system with the evolutionary distance (time) between the two genomes. Our simulation results indicate that there is a wide range of reference genomes at different evolutionary time points that appear to deliver reasonable comparative prediction of human genes. In particular, the evolutionary time between human and mouse generally falls in the region of good performance; however, better accuracy might be achieved with a reference genome further than mouse. To address the second question, we propose several natural comparative measures of conservation for identifying exons and exon boundaries. Finally, we experiment with Bayesian networks for the integration of comparative and compositional evidence.

    Title Direct Molecular Haplotyping of Long-range Genomic Dna with M1-pcr.
    Date July 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for "multiplex" and 1 for "single-copy DNA molecules") that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.

    Title Prediction and Measurement of an Autoregulatory Genetic Module.
    Date July 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The deduction of phenotypic cellular responses from the structure and behavior of complex gene regulatory networks is one of the defining challenges of systems biology. This goal will require a quantitative understanding of the modular components that constitute such networks. We pursued an integrated approach, combining theory and experiment, to analyze and describe the dynamics of an isolated genetic module, an in vivo autoregulatory gene network. As predicted by the model, temperature-induced protein destabilization led to the existence of two expression states, thus elucidating the trademark bistability of the positive feedback-network architecture. After sweeping the temperature, observed population distributions and coefficients of variation were in quantitative agreement with those predicted by a stochastic version of the model. Because model fluctuations originated from small molecule-number effects, the experimental validation underscores the importance of internal noise in gene expression. This work demonstrates that isolated gene networks, coupled with proper quantitative descriptions, can elucidate key properties of functional genetic modules. Such an approach could lead to the modular dissection of naturally occurring gene regulatory networks, the deduction of cellular processes such as differentiation, and the development of engineered cellular control.

    Title Neurological Diseases and Rna-directed Gene Regulation: Prospects for New Diagnostics and Therapy.
    Date July 2003
    Journal Expert Review of Molecular Diagnostics
    Title Designing Isoform-specific Peptide Disruptors of Protein Kinase A Localization.
    Date May 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.

    Title Amino Acid Variant in the Kinase Binding Domain of Dual-specific A Kinase-anchoring Protein 2: a Disease Susceptibility Polymorphism.
    Date May 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity andor mortality. To test this assumption, we compared allele frequencies of 6,500 single-nucleotide polymorphisms (SNPs) located in approximately 5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (d-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an independent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. The IleVal SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound approximately 3-fold weaker to the protein kinase A (PKA)-RIalpha isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of the recombinantly expressed PKA-RIalpha isoform. Our study suggests that alterations in PKA-RIalpha subcellular localization caused by variation in d-AKAP2 may have a negative health prognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health.

    Title Noise in Eukaryotic Gene Expression.
    Date April 2003
    Journal Nature
    Excerpt

    Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

    Title A High-throughput Gene Expression Analysis Technique Using Competitive Pcr and Matrix-assisted Laser Desorption Ionization Time-of-flight Ms.
    Date April 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We report here an approach for gene expression analysis by combining competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. A DNA standard is designed with an artificial single nucleotide polymorphism in the gene of interest. The standard is added to the reverse transcription product before PCR. Subsequently, a base extension reaction is carried out at the single nucleotide polymorphism position, and the products are quantified by matrix-assisted laser desorption ionization time-of-flight MS. The approach is capable of relative and absolute quantification of gene expression; it is extremely sensitive (as few as five copies of DNA were quantified) and highly reproducible. It is also capable of simultaneous quantification of both alleles for heterozygotes and alternatively spliced genes. We have incorporated this technique with the homogeneous Mass Extension system (Sequenom) to create a high-throughput, automated gene expression analysis platform where a few hundred genes from 20-500 different samples can be accurately quantified per day.

    Title Association Testing by Dna Pooling: an Effective Initial Screen.
    Date January 2003
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    With an ever-increasing resource of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. We provide a proof of concept of the use of pooled DNA as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall. We have examined 15 SNPs in the cholesteryl ester transfer protein (CETP) gene, a gene previously demonstrated to be associated with serum high-density lipoprotein cholesterol levels. The SNPs were amplified in two pools of DNA derived from groups of individuals with extremely high and extremely low serum high-density lipoprotein cholesterol levels, respectively. P values <0.05 were obtained for 14 SNPs, supporting the described association. Genotyping of the individual samples showed that the average margin of error in frequency estimate was approximately 4% when pools were used. These findings clearly demonstrate the potential of pooling techniques and their associated technologies as an initial screen in the search for genetic associations.

    Title Automated Genotyping Using the Dna Massarray Technology.
    Date November 2002
    Journal Methods in Molecular Biology (clifton, N.j.)
    Title The Use of Massarray Technology for High Throughput Genotyping.
    Date October 2002
    Journal Advances in Biochemical Engineering/biotechnology
    Excerpt

    This chapter will explore the role of mass spectrometry (MS) as a detection method for genotyping applications and will illustrate how MS evolved from an expert-user-technology to a routine laboratory method in biological sciences. The main focus will be time-of-flight (TOF) based devices and their use for analyzing single-nucleotide-polymorphisms (SNPs, pronounced snips). The first section will describe the evolution of the use of MS in the field of bioanalytical sciences and the protocols used during the early days of bioanalytical MALDI TOF mass spectrometry. The second section will provide an overview on intraspecies sequence diversity and the nature and importance of SNPs for the genomic sciences. This is followed by an exploration of the special and advantageous features of mass spectrometry as the key technology in modern bioanalytical sciences in the third chapter. Finally, the fourth section will describe the MassARRAY technology as an advanced system for automated high-throughput analysis of SNPs.

    Title Base-specific Fragmentation of Amplified 16s Rrna Genes Analyzed by Mass Spectrometry: a Tool for Rapid Bacterial Identification.
    Date June 2002
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria.

    Title High-level Multiplex Dna Amplification.
    Date May 2002
    Journal Antisense & Nucleic Acid Drug Development
    Excerpt

    We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.

    Title Improvement in the Apparent Mass Resolution of Oligonucleotides by Using 12c/14n-enriched Samples.
    Date March 2002
    Journal Analytical Chemistry
    Excerpt

    The apparent mass resolution of oligonucleotides in time-of-flight (TOF) mass spectrometers has been examined. In a reflectron TOF instrument, where the isotopic profile can be completely resolved, the apparent resolution matches the instrument's resolving power. In a linear TOF instrument, unresolved isotopic profiles limit the apparent resolution to much lower values than the actual instrument resolution. By using 12C/14N-enriched oligonucleotides, the apparent resolution can be improved significantly. The isotope enrichment method also enhances the signal-to-noise ratio.

    Title A Streptavidin Mutant Useful for Directed Immobilization on Solid Surfaces.
    Date February 2002
    Journal Bioconjugate Chemistry
    Excerpt

    A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.

    Title Quantitative Mutant Analysis of Viral Quasispecies by Chip-based Matrix-assisted Laser Desorption/ Ionization Time-of-flight Mass Spectrometry.
    Date December 2001
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest.

    Title Dna Microarrays with Stem-loop Dna Probes: Preparation and Applications.
    Date October 2001
    Journal Nucleic Acids Research
    Excerpt

    We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3'-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

    Title Matrix-induced Fragmentation of P3'-n5' Phosphoramidate-containing Dna: High-throughput Maldi-tof Analysis of Genomic Sequence Polymorphisms.
    Date October 2001
    Journal Nucleic Acids Research
    Excerpt

    Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.

    Title Automated Genotyping Using the Dna Massarray Technology.
    Date September 2001
    Journal Methods in Molecular Biology (clifton, N.j.)
    Title High-throughput Development and Characterization of a Genomewide Collection of Gene-based Single Nucleotide Polymorphism Markers by Chip-based Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry.
    Date April 2001
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We describe here a system for the rapid identification, assay development, and characterization of gene-based single nucleotide polymorphisms (SNPs). This system couples informatics tools that mine candidate SNPs from public expressed sequence tag resources and automatically designs assay reagents with detection by a chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. As a proof of concept of this system, a genomewide collection of reagents for 9,115 gene-based SNP genetic markers was rapidly developed and validated. These data provide preliminary insights into patterns of polymorphism in a genomewide collection of gene-based polymorphisms.

    Title Multiplex Allele-specific Target Amplification Based on Pcr Suppression.
    Date April 2001
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We have developed a strategy for multiplex PCR based on PCR suppression. PCR suppression allows DNA target amplification with only one sequence-specific primer per target and a second primer that is common for all targets. Therefore, an n-plex PCR would require only n + 1 primers. We have demonstrated uniform, efficient amplification of targeted sequences in 14-plex PCR. The high specificity of suppression PCR also provides multiplexed amplification with allele specificity. Multiplexed PCR was used to develop assays for genotyping DNA samples from cystic fibrosis-affected individuals. The new approach greatly simplifies primer design, significantly increases the PCR multiplexing level, and decreases the overall primer cost. In addition, this assay is more readily amenable to automation and is therefore suitable for high-throughput genetic diagnostics.

    Title Streptavidin-containing Chimeric Proteins: Design and Production.
    Date February 2001
    Journal Methods in Enzymology
    Title Effects of Saturation Mutagenesis of the Phage Sp6 Promoter on Transcription Activity, Presented by Activity Logos.
    Date May 2000
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A full set of SP6 promoter variants with all possible single substitutions at positions -17 to +5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues -11, -9, -8, -7, and +1 (initiation site). All substitutions at -11, -9, -8, and -7 abolished formation of closed complexes, except for A-8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro. In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at -13, -4, and -2, among others, showed exceptionally higher activities in vivo than in vitro, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A-3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.

    Title New Fluorescent Hydrazide Reagents for the Oxidized 3'-terminus of Rna.
    Date May 2000
    Journal Nucleic Acids Research
    Excerpt

    The synthesis and properties of four new fluorescent reagents capable of forming moderately stable links to the 3' oxidized end of RNA are reported. All are hydrazide derivatives: pyrene butyric acid hydrazide, proflavine monosemicarbazide, proflavine monosuccinic acid hydrazide, and anthracene-9-carboxaldehyde carbohydrazone. In addition, procedures are given for coupling the bifunctional reagent carbohydrazide to the 3' end of RNA. These carbohydrazide adducts can easily be coupled in turn to a wide variety of fluorescent reagents having specificity for aliphatic amino groups, including isothiocyanates and sulfonyl halides. Thus a route exists for the preparation of an enormous variety of 3' fluorescent labeled RNAs. The carbohyrazide adducts are also useful for other synthetic procedures such as preparation of covalent tRNA dimers.

    Title Mass Spectrometry of Single-stranded Restriction Fragments Captured by an Undigested Complementary Sequence.
    Date April 2000
    Journal Nucleic Acids Research
    Excerpt

    In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin-streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.

    Title Oligonucleotide Immobilization on Micropatterned Streptavidin Surfaces.
    Date April 2000
    Journal Nucleic Acids Research
    Excerpt

    We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n(-3/4).

    Title Pharmacogenetics Becomes Pharmacogenomics: Wake Up and Get Ready.
    Date March 2000
    Journal Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology
    Title Construction of a Genetic Toggle Switch in Escherichia Coli.
    Date February 2000
    Journal Nature
    Excerpt

    It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements. These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator. However, these behaviours have not been demonstrated in networks of non-specialized regulatory components. Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability. The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network. It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold. As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy.

    Title Biotechnology in the 21st Century.
    Date January 2000
    Journal Trends in Biotechnology
    Excerpt

    Although the future is unpredictable, it is highly likely that biotechnology will play a much more visible and significant role in the 21st century than it did in the 20th century. The number and kinds of drugs provided by biotechnology will expand markedly and biotechnology will stand at the center of the oncoming revolution in bioinformatics.

    Title In Silico Detection of Control Signals: Mrna 3'-end-processing Sequences in Diverse Species.
    Date January 2000
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We have investigated mRNA 3'-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3'-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3'-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3'-end-processing signals will require more than consensus sequence identification.

    Title Chip-based Genotyping by Mass Spectrometry.
    Date October 1999
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-microl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.

    Title Mass Spectrometry of Nucleic Acids.
    Date September 1999
    Journal Clinical Chemistry
    Title Differential Sequencing with Mass Spectrometry.
    Date May 1999
    Journal Genetic Analysis : Biomolecular Engineering
    Excerpt

    Differential or genetic sequencing requires searching sample DNA for variations with respect to a reference sequence. Conventional detection techniques are too labor and cost expensive for use in diagnostic applications, therefore new technologies will be required. Measurement techniques based on mass spectrometry (MS) possess the potential for high-throughput, high fidelity measurement of sequence variation. Unambiguous detection of polymorphic sequences has been demonstrated, even in heterozygous samples. Automated reproducible measurements of microscopic arrays of samples will enable the high-throughput detection required for large-scale applications. Computational simulation and analysis of experimental parameters prior to experimentation will provide the optimization necessary for development of robust, reproducible measurements.

    Title Gait Disorders.
    Date March 1999
    Journal Clinics in Podiatric Medicine and Surgery
    Excerpt

    Normal gait requires the dynamic integration of central and peripheral nervous systems acting on an intact musculoskeletal framework. A number of specific disease processes, as well as aging, may compromise this interaction. Despite the complexity of human gait, most common gait disorders can be identified by the experienced clinician, using the fundamental tools of history and physical examination.

    Title Genomic Detection of New Yeast Pre-mrna 3'-end-processing Signals.
    Date March 1999
    Journal Nucleic Acids Research
    Excerpt

    To investigate Saccharomyces cerevisiae 3'-end-processing signals, a set of 1352 unique pre-mRNA 3'-end-processing sites, corresponding to 861 different genes, was identified by alignment of expressed sequence tag sequences with the complete yeast genome. Nucleotide word frequencies in the vicinity of the cleavage sites were analyzed to reveal the signal element features. In addition to previously recognized processing signals, two previously uncharacterized components of the 3'-end-processing signal sequence were discovered, specifically a predominance of U-rich sequences located on either side of the cleavage site. One of these, the downstream U-rich signal, provides a further link between the 3'-end-processing mechanisms of yeast and higher eukaryotes. Analysis of the complete set of 3'-end-processing sites by means of a discrimination function supports a 'contextual' model in which the sum total effectiveness of the signals in all four elements determines whether or not processing occurs.

    Title Genetic Engineering of Streptavidin, a Versatile Affinity Tag.
    Date December 1998
    Journal Journal of Chromatography. B, Biomedical Sciences and Applications
    Excerpt

    Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of streptavidin and to expand the application of streptavidin as an affinity tag. In this review, we describe several genetically engineered streptavidin variants, which include a streptavidin with a reduced biotin-binding affinity, a dimeric streptavidin, and a fusion protein between streptavidin and protein A, along with their potential applications in biological science.

    Title A Streptavidin Mutant with Altered Ligand-binding Specificity.
    Date December 1998
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 x 10(6) M-1, two orders of magnitude higher than that for biotin (1 x 10(4) M-1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 x 10(4) M-1) was lower than predicted (2.9 x 10(5) M-1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.

    Title Massive Attack on High-throughput Biology.
    Date October 1998
    Journal Nature Genetics
    Title Lighting Up Hybridization.
    Date July 1998
    Journal Nature Biotechnology
    Title A Strategy for Rapid and Efficient Dna Sequencing by Mass Spectrometry.
    Date July 1998
    Journal Nature Biotechnology
    Excerpt

    Two methods of solid-phase Sanger DNA sequencing followed by detection with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are demonstrated. In one method, sequencing ladders generated on an immobilized synthetic template were resolved up to the 63-mer including the primer. Detection sensitivity and resolution were sufficient for sequence analysis in the given range. This approach is particularly suitable for comparative (diagnostic) DNA sequencing. A second method that has the potential for high throughput de novo DNA sequencing is also presented; it uses immobilized duplex probes with five-base single-stranded overhangs to capture an unknown DNA template serving as primers for Sanger DNA sequencing. The power of mass spectrometry is demonstrated not only by its very high speed, but also by its ability to identify sequences that are not readable using gel electrophoresis.

    Title Streptavidins with Intersubunit Crosslinks Have Enhanced Stability.
    Date July 1998
    Journal Nature Biotechnology
    Excerpt

    Natural tetrameric streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural streptavidin. The first and second streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core streptavidin after heat treatment. All of these crosslinked streptavidins retained bound biotin more stably than natural core streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of streptavidin.

    Title Sequencing Exons 5 to 8 of the P53 Gene by Maldi-tof Mass Spectrometry.
    Date May 1998
    Journal Nature Biotechnology
    Excerpt

    Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.

    Title How Will the Human Genome Project Improve Our Quality of Life?
    Date May 1998
    Journal Nature Biotechnology
    Title Probing the Genetic Population Structure of Trypanosoma Cruzi with Polymorphic Microsatellites.
    Date April 1998
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.

    Title Advances in Dna Diagnostics.
    Date April 1998
    Journal Current Opinion in Biotechnology
    Excerpt

    The key advances in DNA diagnostics during the past year are techniques which will lead to advanced throughput without sacrificing sensitivity: miniaturization of samples to reduce material cost and preparation time, and parallelization through use of measurement arrays. The most promising gains have come in the areas of DNA arrays and mass spectrometry, where differential sequencing measurements are now possible.

    Title Principal Transcription Sigma Factors of Pseudomonas Putida Strains Mt-2 and G1 Are Significantly Different.
    Date February 1998
    Journal Gene
    Excerpt

    The rpoD gene coding for the primary transcription sigma factor, sigma70, and its entire operon were cloned from strain mt-2 of the purple soil bacterium Pseudomonas putida. Comparison of the deduced amino acid sequence of Ppmt-2 sigma70 with that of sigma70 from P. putida strain G1 shows that the two proteins differ in their primary structure, molecular weight, and isoelectric point. The significance of this difference is discussed in terms of bacterial taxonomy and transcription regulation.

    Title Instrumentation in Molecular Biomedical Diagnostics: an Overview.
    Date December 1997
    Journal Genetic Analysis : Biomolecular Engineering
    Title Deoxyribonucleic Acids As Unique Markers in Molecular Detection.
    Date December 1997
    Journal Genetic Analysis : Biomolecular Engineering
    Title Cloning and Analysis of the Dnag Gene Encoding Pseudomonas Putida Dna Primase.
    Date August 1997
    Journal Biochimica Et Biophysica Acta
    Excerpt

    The dnaG gene coding for primase, a key enzyme in DNA replication, has been isolated from chromosomal DNA of the soil bacterium Pseudomonas putida. It maps within the putative MMS operon, between the rpsU and rpoD genes. Comparison of the deduced amino acid sequence of P. putida DnaG with sequences of other known bacterial primases reveals the presence of a possible regulatory region which would be unique to pseudomonads. The analysis of nucleotide sequence suggests that stable folding of the dnaG mRNA may significantly contribute to the low level of its expression within a cell.

    Title Engineering Subunit Association of Multisubunit Proteins: a Dimeric Streptavidin.
    Date July 1997
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A dimeric streptavidin has been designed by molecular modeling using effective binding free energy calculations that decompose the binding free energy into electrostatic, desolvation, and side chain entropy loss terms. A histidine-127 --> aspartic acid (H127D) mutation was sufficient to introduce electrostatic repulsion between subunits that prevents the formation of the natural tetramer. However, the high hydrophobicity of the dimer-dimer interface, which would be exposed to solvent in a dimeric streptavidin, suggests that the resulting molecule would have very low solubility in aqueous media. In agreement with the calculations, a streptavidin containing the H127D mutation formed insoluble aggregates. Thus, the major design goal was to reduce the hydrophobicity of the dimer-dimer interface while maintaining the fundamental structure. Free energy calculations suggested that the hydrophobicity of the dimer-dimer interface could be reduced significantly by deleting a loop from G113 through W120 that should have no apparent contact with biotin in a dimeric molecule. The resulting protein, containing both the H127D mutation and the loop deletion, formed a soluble dimeric streptavidin in the presence of biotin.

    Title Expression and Purification of Recombinant Streptavidin-containing Chimeric Proteins.
    Date June 1997
    Journal Methods in Molecular Biology (clifton, N.j.)
    Title Interaction of Biotin with Streptavidin. Thermostability and Conformational Changes Upon Binding.
    Date May 1997
    Journal The Journal of Biological Chemistry
    Excerpt

    The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.

    Title In Situ Detection of Tandem Dna Repeat Length.
    Date April 1997
    Journal Genetic Analysis : Biomolecular Engineering
    Excerpt

    A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats known lengths. Single-stranded loops structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase.

    Title A New Approach for Containment of Microorganisms: Dual Control of Streptavidin Expression by Antisense Rna and the T7 Transcription System.
    Date March 1997
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal Streptomyces avidinii streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group, D-biotin. The entire construct was designed to allow the soil bacterium Pseudomonas putida to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.

    Title Sequencing Double-stranded Dna by Strand Displacement.
    Date March 1997
    Journal Nucleic Acids Research
    Excerpt

    Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequenced without subcloning, purification or denaturation.

    Title The Development of Microfabricated Arrays for Dna Sequencing and Analysis.
    Date February 1997
    Journal Trends in Biotechnology
    Excerpt

    Microfabricated arrays of immobilized oligodeoxynucleotide probes are proving to be a powerful tool for rapidly generating sequence data via hybridization. These arrays are made either by immobilization of the probe post-synthetically, or by in situ synthesis of the probe. Hybridization of the target is easily achieved on the arrays, with analysis proceeding either by direct detection, or through enzyme-mediated detection; analysis of the hybridization pattern yields sequence information about the target. Such facile and rapid data acquisition will assist the challenging task of sequencing the human genome, and also will lead to a new generation of diagnostic assays.

    Title Molecular Engineering of Streptavidin.
    Date January 1997
    Journal Annals of the New York Academy of Sciences
    Title Efficient Preparation of Short Dna Sequence Ladders Potentially Suitable for Maldi-tof Dna Sequencing.
    Date August 1996
    Journal Genetic Analysis : Biomolecular Engineering
    Excerpt

    Duplex probes with five-base single-stranded overhangs were developed for positional sequencing by hybridization [Broude et al., Proc Natl Acad Sci USA 91:3072-3076, 1994]. The partially duplex probes can be employed to capture single-stranded oligonucleotide targets and form primer-template complexes. Recently we showed that partially duplex probes can prime Sanger sequencing reactions on immobilized, but non-ligated long single-stranded targets (approximately 500 nucleotide) [Fu et al., Proc Natl Acad Sci, in press]. Here immobilized, non-ligated partially duplex probes were used to capture and sequence short single-stranded targets. This strategy is capable of rapidly preparing large numbers of samples for future mass spectrometric DNA sequencing.

    Title Rna Loop Structure Prediction Via Bond Scaling and Relaxation.
    Date July 1996
    Journal Biopolymers
    Excerpt

    We have developed a method for predicting the structure of small RNA loops that can be used to augment already existing RNA modeling techniques. The method requires no input constraints on loop configuration other than end-to-end distance. Initial loop structures are generated by randomizing the torsion angles, beginning at one end of the polynucleotide chain and correlating each successive angle with the previous. The bond lengths of these structures are then scaled to fit within the known end constraints and the equilibrium bond lengths of the potential energy function are scaled accordingly. Through a series of rescaling and minimization steps the structures are allowed to relax to lower energy configurations with standard bond lengths and reduced van der Waals clashes. This algorithm has been tested on the variable loops of yeast tRNA-Asp and yeast tRNA-Phe, as well as the sarcin-ricin tetraloop and the anticodon loop of yeast tRNA-Phe. The results indicate good correlation between potential energy and the loop structure predictions that are closest to the variable loop crystal structures, but poorer correlation for the more isolated stem loops. The number of stacking interactions has proven to be a good objective measure of the best loop predictions. Selecting on the basis of energy and stacking, we obtain two structures with 0.65 and 0.75 A all-atom rms deviations (RMSD) from the crystal structure for the tRNA-Asp variable loop. The best structure prediction for the tRNA-Phe variable loop has an all-atom RMSD of 2.2 A and a backbone RMSD of 1.6 A, with a single base responsible for most of the deviation. For the sarcin-ricin loop from 28S ribosomal RNA, the predicted structure's all-atom RMSD from the nmr structure is 1.0 A. We obtain a 1.8 A RMSD structure for the tRNA-Phe anticodon loop.

    Title Comparative Properties of a Technetium-99m-labeled Single-stranded Natural Dna and a Phosphorothioate Derivative in Vitro and in Mice.
    Date February 1996
    Journal The Journal of Pharmacology and Experimental Therapeutics
    Excerpt

    Oligonucleotides, particularly single stranded, may ultimately be of considerable use as radiopharmaceuticals. We have compared a synthetic 22-base single-stranded phosphodiester DNA with its phosphorothioate analog after both were radiolabeled with 99mTc via the hydrazino nicotinamide chelator. Whole body clearance of the label in mice was much slower when introduced on the phosphorothioate (30% vs. 75% clearance at 6 hr) because of immediate and persistent accumulation in liver (47% vs. 2% injected dose/g at 4 hr). The label in both cases was present in urine primarily on low molecular weight catabolites. High-performance liquid chromatography analysis of 37 degrees C serum incubates showed serum protein binding of 99mTc in both cases (about 100% bound at 24 hr) but to different proteins. Different behavior with respect to protein binding was also observed in the analysis of liver and kidney homogenates: the phosphodiester label was almost quantitatively converted to lower molecular weight catabolites after only 15 min, whereas the phosphorothioate label was primarily on proteins. The rapid digestion of the phosphodiester by nucleases was not observed, probably because protein binding of the labeled oligonucleotides stabilized against degradation. Thus the phosphodiester DNA may be the preferred 99mTc-labeled oligonucleotide in certain circumstances to avoid the high and persistent liver uptake observed with the phosphorothioate DNA.

    Title Technetium-99m Labeling of Dna Oligonucleotides.
    Date January 1996
    Journal Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
    Excerpt

    Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99mTc can be developed. METHODS: To radiolabel DNA with 99mTc, we have used the hydrazino nicotinamide (SHNH) moiety developed elsewhere. The diethylenetriaminepentacetic acid (DTPA) chelate was used to label DNA with 111In for comparison. Complementary 22-base, single-stranded oligonucleotides were obtained, each with a primary amine attached to either 3' or 5' end with a biotin moiety on the opposite end. The DNA was conjugated with SHNH by a N-hydroxysuccinimide derivative with DTPA by the cyclic anhydride. RESULTS: Reversed-phase HPLC analysis showed that essentially complete conjugation was achieved in both cases. The purified SHNH-DNA was radiolabeled with 99mTc by transchelation from glucoheptonate at labeling efficiencies of up to 60% and DTPA-DNA with 111In acetate at up to 100% efficiency. After labeling, the ability of the DNAs to bind to streptavidin through the biotin moieties and to hybridize with their complementary DNA in saline was retained for both radiolabels as determined by size-exclusion HPLC analysis. HPLC radiochromatograms of serum incubates showed a shift to 99mTc, but not 111In, to a high molecular weight, strongly suggesting serum protein binding in the former case only. Low-molecular weight degradation products were seen with 111In, but not with 99mTc and may be related to the use of phosphodiester-linked oligonucleotides. As a further measure of label stability, the DNAS were bound to streptavidin-conjugated magnetic beads and incubated in fresh 37 degrees C human serum. Less than 4% of 99mTc and 14% of 111In was lost in 24 hr. CONCLUSION: Amino-modified, single-stranded DNA can be stably radiolabeled with 99mTc by the SHNH moiety without loss of function.

    Title Recombinant Core Streptavidins. A Minimum-sized Core Streptavidin Has Enhanced Structural Stability and Higher Accessibility to Biotinylated Macromolecules.
    Date January 1996
    Journal The Journal of Biological Chemistry
    Excerpt

    Two recombinant core streptavidins were designed and characterized to understand the role of the terminal sequences, present in naturally truncated core streptavidins, in the properties of streptavidin. One recombinant core streptavidin, Stv-25, has an amino acid sequence very similar to natural core streptavidins. The other recombinant molecule, Stv-13, has further truncation of the terminal residues and consists essentially of only the beta-barrel structure characteristic of streptavidin. These recombinant core streptavidins are tetrameric and bind four biotins/molecule, as does natural streptavidin. The solubility characteristics of Stv-13, determined by varying the concentration of ammonium sulfate or ethanol, were almost the same as those of Stv-25 and natural core streptavidin. However, Stv-13 showed an enhanced structural stability compared with Stv-25 and natural core streptavidin. For example, Stv-13 retained greater than 80% of its biotin binding ability after incubation in 6 M guanidine hydrochloride at pH 1.5, under which conditions, Stv-25 and natural core streptavidin retained only about 20% of their biotin binding ability. In addition, Stv-13 showed higher accessibility to biotinylated DNA than natural core streptavidin. Apparently, the terminal regions, present on the surface of natural core streptavidin, spatially hinder biotinylated macromolecules from approaching the biotin binding sites.

    Title Large Scale Isolation of Expression Vector Cassette by Magnetic Triple Helix Affinity Capture.
    Date November 1995
    Journal Nucleic Acids Research
    Title A Dna Sequencing Strategy That Requires Only Five Bases of Known Terminal Sequence for Priming.
    Date November 1995
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

    Title Matrix-assisted Laser Desorption/ionization Mass Spectrometry of Immobilized Duplex Dna Probes.
    Date October 1995
    Journal Nucleic Acids Research
    Excerpt

    Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry was used to analyze short DNA duplex probes with one strand immobilized on solid supports (straptavidin-coated magnetic beads or controlled pore glass beads). Only the non-immobilized strand could be detected. Partial denaturation was found when the duplex probes were mixed with 3-hydroxypicolinic acid, ammonium citrate matrix. The strategy has several applications, such as fast DNA sequence analysis and DNA diagnostics.

    Title Intersubunit Contacts Made by Tryptophan 120 with Biotin Are Essential for Both Strong Biotin Binding and Biotin-induced Tighter Subunit Association of Streptavidin.
    Date May 1995
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    In natural streptavidin, tryptophan 120 of each subunit makes contacts with the biotin bound by an adjacent subunit through the dimer-dimer interface. To understand quantitatively the role of tryptophan 120 and its intersubunit communication in the properties of streptavidin, a streptavidin mutant in which tryptophan 120 is converted to phenylalanine was produced and characterized. The streptavidin mutant forms a tetrameric molecule and binds one biotin per subunit, as does natural streptavidin, indicating that the mutation of tryptophan 120 to phenylalanine has no significant effect on the basic properties of streptavidin. However, its biotin-binding affinity was reduced substantially, to approximately 10(8) M-1, indicating that the contact made by tryptophan 120 to biotin has a considerable contribution to the extremely tight biotin binding by streptavidin. The mutant retained bound biotin over a wide pH range or with the addition of urea up to 6 M at neutral pH. However, bound biotin was efficiently released by the addition of excess free biotin due, presumably, to exchange reactions. Electrophoretic analysis revealed that the intersubunit contact made by tryptophan 120 to biotin through the dimer-dimer interface is the major interaction responsible for the biotin-induced, tighter subunit association of streptavidin. In addition, the mutant has weaker subunit association than natural streptavidin even in the absence of biotin, indicating that tryptophan 120 also contributes to the subunit association of tetramers in the absence of biotin.

    Title Recombinant Metallothionein-conjugated Streptavidin Labeled with 188re and 99mtc.
    Date May 1995
    Journal Bioconjugate Chemistry
    Excerpt

    Consideration is now being given to the use of avidin (or streptavidin) and biotin for radiotherapy of tumor. Accordingly, the goal of this study was to radiolabel a mouse metallothionein-streptavidin fusion protein with 188Re and to compare its properties to those of the same fusion protein radiolabeled with 99mTc. A recombinant metallothionein-streptavidin fusion protein was radiolabeled by transchelation with 99mTc- and 188Re-glucoheptonate. Labeling efficiency, which was not optimized for either radionuclide, was approximately 60% for 99mTc and 20% for 188Re. Radiochemical purity was demonstrated by size exclusion HPLC both by nearly quantitative shifts of the 188Re label to higher molecular weight upon the addition of biotinylated antibody and by the absence of a shift with biotinsaturated 188Re-metallothionein-streptavidin. Stability of the labels in 37 degrees C serum was evaluated by comparing the HPLC radiochromatograms of serum samples both before and after the addition of biotinylated antibody. The 188Re label behaved like 99mTc in that the same peaks were evident, including one prominent peak due to labeled cysteine. Recoveries during HPLC analysis of serum samples showed that oxidation rates to perrhenate and pertechnetate were identical. However, instability to cysteine challenge was greater for 188Re; for example, the loss of label to cysteine after 24 h under one set of conditions was 41% for 188Re and 22% with 99mTc. Analysis by HPLC of liver and kidney homogenates from mice administered the labeled antibodies were qualitatively and, in large measure, quantitatively independent of label. Biodistributions at 5 h in normal mice were statistically identical between the two labels in blood and in most tissues. In conclusion, streptavidin may be radiolabeled with radiorhenium using recombinant mouse metallothionein as a bifunctional chelator, and under one set of labeling conditions at least, 188Re showed similar in vitro and in vivo behavior to that of 99mTc labeled to the same fusion protein.

    Title Oligonucleotide-directed Self-assembly of Proteins: Semisynthetic Dna--streptavidin Hybrid Molecules As Connectors for the Generation of Macroscopic Arrays and the Construction of Supramolecular Bioconjugates.
    Date February 1995
    Journal Nucleic Acids Research
    Excerpt

    Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.

    Title Top-down Construction of an Ordered Schizosaccharomyces Pombe Cosmid Library.
    Date June 1994
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A very rapid and efficient method for sorting and ordering large numbers of clones is presented. This top-down mapping approach divides the entire ordering problem into many smaller tasks and analyzes in parallel a gridded membrane array of clones by hybridization with probe pools. The strategy was tested on a 15-fold-coverage Schizosaccharomyces pombe cosmid library. About 1600 clones were assigned to chromosomes and to regions defined by the Not I and Sfi I restriction maps. Then, the clones were ordered into 20 contigs, which is consistent with statistical expectations for the degree of genome coverage used. The parallel ordering of clones and the computer-based analysis of digitized images make this approach very efficient; it is about 8-fold faster than existing methods. Only 61 hybridizations were needed to order 1600 clones.

    Title Enhanced Dna Sequencing by Hybridization.
    Date May 1994
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    An enhanced version of DNA sequencing by hybridization (SBH), termed positional SBH (PSBH), has been developed. PSBH uses duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target should provide enhanced stringency in distinguishing perfectly matched 3' sequences. A second enhancement is the use of enzyme-catalyzed steps, instead of pure physical hybridization. The feasibility of this scheme has been investigated using biotinylated duplex probes containing single-stranded 5-base 3' overhangs, immobilized on streptavidin-coated magnetic beads. Ligation of a single-stranded target, hybridized to the single-stranded region of the duplex probes, provided enhanced discrimination of perfectly matched targets from those containing mismatches. In distinction to the serious complications caused by base composition effects in ordinary SBH, there was little effect of base composition in PSBH. The hardest mismatch to discriminate was the one furthest from the phosphodiester bond formed by ligation. However, mismatches in this position were efficiently discriminated by 3' extension of the duplex probe using a template-dependent DNA polymerase. These results demonstrate that PSBH offers considerable promise to facilitate actual implementations of SBH.

    Title Dna Sequence Analysis of Human Chromosome 21 Noti Linking Clones.
    Date February 1994
    Journal Genomics
    Excerpt

    Portions of 16 chromosome 21 NotI linking clones were sequenced. These linking clone sequences represent sequence-tagged restriction sites that are potentially useful for finding genes and for finer genome mapping and sequencing. All of the clones were G+C rich (54 to 83%). CpG and GpC dinucleotide frequencies were very close to the expected values based on base composition and were very similar in 15 of the clones. Most of the NotI linking clones were derived from CpG islands, which are often associated with genes. Five NotI linking clones had a high potential for coding regions; 7 additional clones may also contain coding regions. The NotI linking clones had many short homologous regions, but no extensive homologies either with each other or with GenBank sequences.

    Title Effect of Cd4 Engagement on Cd4-t Cell Receptor Complexes.
    Date January 1994
    Journal Cellular Immunology
    Excerpt

    To detect the presence of CD4-T cell receptor (TCR) complexes, we previously developed a flow cytometric method for measuring singlet-singlet energy transfer on human T cells labeled with fluorescein isothiocyanate-conjugated anti-CD4 and tetramethylrhodamine isothiocyanate-conjugated anti-TCR. Using the same procedure, we have now studied changes in the expression of CD4, TCR, and CD4-TCR complexes following CD4 engagement. Ligation of the D3 domain with OKT4, or the D1 domain with anti-Leu3a, induced CD4 and TCR down-regulation, while ligation of the D1 domain with gp120 did not. OKT4 caused a transient decrease in CD4-TCR association over 1 hr at 37 degrees C, while anti-Leu3a caused a steady-state decrease. In contrast, gp120 decreased CD4-TCR association mainly at 0 degrees C, rather than at 37 degrees C. Such alteration in CD4-TCR assembly may underly anti-Leu3a- and gp120-mediated inhibition of T cell antigen recognition and account for the negative effect of CD4 ligation on TCR-triggered responses.

    Title The 0.7 to 3.3 Megabase Chromosomes from Candida, Kluyveromyces, and Pichia Provide Accurate Size Standards for Pulsed Field Gel Electrophoresis.
    Date June 1993
    Journal Electrophoresis
    Excerpt

    Pulsed field gel electrophoresis was used to size intact chromosomal DNAs from Candida albicans, Kluyveromyces lactis, Pichia scolyti, and Pichia mississippiensis by optimization methods using, as size standards, concatenated bacteriophage lambda DNA, and intact and NotI digestion products of Schizosaccharomyces pombe chromosomal DNAs. These newly sized fungal DNAs can now serve as convenient and accurate size standards for DNA molecules between 0.7 and 3.3 megabases (Mb). These size standards are valid over a wide range of different electrophoretic conditions.

    Title Atomic Force Microscopy of Biochemically Tagged Dna.
    Date May 1993
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Small fragments of DNA of known length were made with the polymerase chain reaction. These fragments had biotin molecules covalently attached at their ends. They were subsequently labeled with a chimeric protein fusion between streptavidin and two immunoglobulin G-binding domains of staphylococcal protein A. This tetrameric species was expected to bind up to four DNA molecules via their attached biotin moieties. The DNA-protein complex was deposited on mica and imaged with an atomic force microscope. The images revealed the protein chimera at the expected location at the ends of the strands of DNA as well as the expected dimers, trimers, and tetramers of DNA bound to a single protein.

    Title Pcr Amplification of Megabase Dna with Tagged Random Primers (t-pcr).
    Date May 1993
    Journal Nucleic Acids Research
    Title Cloning and Characterization of Eagi Yacs from Human Chromosome 21.
    Date March 1993
    Journal Genomics
    Excerpt

    Yeast artificial chromosomes (YACs) were made from a total EagI digest of DNA from a mouse-human chromosome 21 hybrid cell line. Approximately 3750 YACs, corresponding to 75-125 human YACs, with an average size of approximately 100 kb were recovered. Southern hybridization indicates that the chimera frequency in this library may be less than 3%. Thirty-four of the human EagI YACs were regionally assigned by a number of methods. Some YACs were regionally assigned to one of six chromosome regions by hybridization of Alu-PCR products from the YAC against Alu-PCR-amplified DNA from a panel of hybrid cell lines that contain various parts of chromosome 21. Additional YACs were regionally assigned by fluorescence in situ hybridization using either biotinylated Alu-PCR products or yeast genomic DNA from the YAC-containing strains as probes. The regionally assigned EagI YACs are located preferentially in two regions of the chromosome: near the q telomere and in the p-arm ribosomal gene region.

    Title A Streptavidin Mutant Containing a Cysteine Stretch That Facilitates Production of a Variety of Specific Streptavidin Conjugates.
    Date March 1993
    Journal Bio/technology (nature Publishing Company)
    Excerpt

    The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites. A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity. Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer. Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry. In this manner, S-[14C]carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared. These conjugates contain almost twenty [14C]carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive. More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.

    Title Affinity Capture Electrophoresis for Sequence-specific Dna Purification.
    Date February 1993
    Journal Genetic Analysis, Techniques and Applications
    Excerpt

    A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecule and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.

    Title Immuno-pcr: Very Sensitive Antigen Detection by Means of Specific Antibody-dna Conjugates.
    Date November 1992
    Journal Science (new York, N.y.)
    Excerpt

    An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 x 10(-22) moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately x 10(5)) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.

    Title Report on the Sequencing by Hybridization Workshop.
    Date September 1992
    Journal Genomics
    Title Triplex Affinity Capture of a Single Copy Clone from a Yeast Genomic Library.
    Date August 1992
    Journal Nucleic Acids Research
    Title Psoralen Damage-induced Plasmid Recombination in Saccharomyces Cerevisiae: Dependence on Rad1 and Rad52.
    Date June 1992
    Journal Mutation Research
    Excerpt

    Photoreaction with psoralen, a DNA-crosslinking reagent, induces mitotic recombination in the yeast Saccharomyces cerevisiae. Psoralen damage-induced recombination was studied with non-replicating plasmids, which transform yeast cells by undergoing recombination events with chromosomal DNA. When plasmid DNA was photoreacted with psoralen in vitro and transformed into yeast cells, transformation was stimulated by psoralen modification in a dose-dependent manner. The stimulation by psoralen damage requires RAD52 gene function and is partially dependent on RAD1. Analysis of transformants indicates that plasmid integration occurs at the homologous chromosomal loci. Multiple tandem integrations are common in repair-proficient cells, with more than 20 copies of integrated plasmid seen in some transformants. Multiple integration depends on RAD1 function; only 9% of rad1 transformants, compared to 80% of RAD transformants, contained multiple plasmid copies, while 52% of the rad1 transformants were produced by gene conversion.

    Title A Contiguous Not I Restriction Map of Band Q22.3 of Human Chromosome 21.
    Date May 1992
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A contiguous high-resolution NotI restriction map of the distal region of the long arm of human chromosome 21 was constructed by three strategies: linking clones to identify adjacent pieces of DNA, partial digestion to identify neighboring fragments, and cell line polymorphisms to prove identity or adjacency of DNA fragments. Twenty-nine single-copy DNA probes and five linking clone probes were used to determine the order of 30 Not I fragments, covering 10 megabases of DNA in band q22.3. Smaller Not I fragments occur preferentially in this region, suggesting that band q22.3 is unusually rich in genes, since Not I sites occur almost exclusively in CpG islands. Comparison of the physical map and genetic maps in this region reveals a 10-fold higher than average recombination frequency.

    Title Budgeting the Genome.
    Date April 1992
    Journal Trends in Biotechnology
    Title A Streptavidin-metallothionein Chimera That Allows Specific Labeling of Biological Materials with Many Different Heavy Metal Ions.
    Date April 1992
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We have designed a streptavidin-metallothionein chimeric protein in which the streptavidin moiety provides a means of binding the metallothionein moiety tightly to specific biological targets. A gene fusion of streptavidin with mouse metallothionein I was efficiently expressed in Escherichia coli, and the expressed chimeric protein was purified to homogeneity by a simple procedure. The purified chimera, consisting of four identical subunits, bound one biotin and approximately seven Cd2+ ions per subunit (19.5 kDa). This indicates that both the streptavidin and the metallothionein moieties are fully functional. The high binding affinity of the chimera both for biotin and for heavy metal ions allows the specific labeling or conjugation of any biological material containing unhindered biotin with a variety of different heavy metal ions and their isotopes, thereby opening the way for simultaneous assay systems for a large number of biological targets.

    Title The Effect of Dna Concentration on Mobility in Pulsed Field Gel Electrophoresis.
    Date April 1992
    Journal Nucleic Acids Research
    Excerpt

    The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA concentrations of 200 micrograms/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800 micrograms/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 micrograms/ml (10(7) diploid human cells/ml agarose insert).

    Title Genome Sequencing Conference. Iii: Evolution and Progress.
    Date March 1992
    Journal Genomics
    Title Distribution of Interspersed Repeats (alu and Kpn) on Noti Restriction Fragments of Human Chromosome 21.
    Date March 1992
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Interspersed repeated sequences (Alu and Kpn) were used as probes to detect a set of Not I restriction fragments of human chromosome 21 from the hybrid cell line WAV17. Forty different Not I fragments, ranging in size from less than 0.05 megabase (Mb) to 7.0 Mb, were identified. The total length of these fragments was 47.3 Mb. This length provides an estimate of the minimum size of the chromosome and a minimum number of fragments to be ordered to create a complete restriction map. The average length Not I fragment is 1.2 Mb. Alu and Kpn fragments are not always coincident: a 2.9-Mb fragment is detected with Kpn but not with Alu, and 13 fragments, ranging from less than 0.05 Mb to 5.6 Mb, are detected with Alu but not with Kpn; the 26 remaining fragments, covering 75% (35.3 Mb) of the total length, are detected with both repetitive probes. The presence of so many noncoincident fragments and the high variation of the hybridization signal intensities of the fragments suggest a very nonuniform distribution of Kpn and Alu repeats.

    Title Call for a Worldwide Survey of Human Genetic Diversity: a Vanishing Opportunity for the Human Genome Project.
    Date February 1992
    Journal Genomics
    Title Sequence-specific Dna Purification by Triplex Affinity Capture.
    Date February 1992
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    A DNA isolation procedure was developed by using triple-helix formation and magnetic separation. In this procedure, target DNA is captured by a biotinylated oligonucleotide via intermolecular triplex formation, bound to streptavidin-coated magnetic beads, and recovered in double-stranded form by elution with a mild alkaline buffer that destabilizes the triple helix. The effectiveness of the procedure was demonstrated by a model experiment with an artificially reconstructed library and, also, by the isolation of (dT-dC)n.(dG-dA)n dinucleotide repeats from a human genomic library. This procedure provides a prototype for other triplex-mediated DNA isolation technologies.

    Title Long-distance Restriction Mapping of the Proximal Long Arm of Human Chromosome 21 with Not I Linking Clones.
    Date February 1992
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

    Title A Streptavidin-protein A Chimera That Allows One-step Production of a Variety of Specific Antibody Conjugates.
    Date February 1992
    Journal Bio/technology (nature Publishing Company)
    Excerpt

    We have expressed a gene fusion of streptavidin and the two immunoglobulin G (IgG)-binding domains of protein A in Escherichia coli. The purified chimeric protein had full biotin-binding ability, and bound one IgG molecule per subunit (31.4 kD). The affinity of the chimeric protein both for biotin and IgG can be used to provide antibody molecules with additional recognition capabilities. For example, the chimera will allow any IgG molecule to be used for sensitive biotin-linked detection systems without the need for chemical modification.

    Title Construction and Characterization of a Noti Linking Library of Human Chromosome 21.
    Date October 1991
    Journal Genomics
    Excerpt

    Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.

    Title Secondary Pulsed Field Gel Electrophoresis: a New Method for Faster Separation of Larger Dna Molecules.
    Date June 1991
    Journal Nucleic Acids Research
    Excerpt

    A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols.

    Title Giardia Lamblia: Haploid Genome Size Determined by Pulsed Field Gel Electrophoresis is Less Than 12 Mb.
    Date June 1991
    Journal Nucleic Acids Research
    Excerpt

    Previous estimates of the size of the Giardia lamblia genome have ranged from 30 to 80 million base pairs (Mb), based on DNA renaturation kinetics. This is much larger than the sum of the sizes of the 4 to 5 chromosomal DNAs seen in typical pulsed field gel electrophoretic analyses. One possible explanation is that each visible chromosomal DNA consists of several unresolved DNA species. To examine this we have performed quantitative densitometry of ethidium stained chromosomal DNAs and Notl genomic digests. We have also examined the distribution of rDNA on Notl genomic fragments. All of our results suggests that the true genome size is 10.6 to 11.9 Mb. It is conceivable that the previous larger estimates may be distorted by impurities in the DNA preparations used.

    Title Expression Vectors for Streptavidin-containing Chimeric Proteins.
    Date June 1991
    Journal Biochemical and Biophysical Research Communications
    Excerpt

    We have constructed expression vectors for streptavidin-containing chimeric proteins. These vectors carry the DNA sequence corresponding to the core region of the streptavidin molecule, and have several unique cloning sites which facilitate construction of gene fusions of streptavidin with a target protein. A chimeric protein of streptavidin and the target protein should be expressible in Escherichia coli by using the T7 expression system. Because of the strong and specific biotin-binding affinity of the streptavidin moiety, such streptavidin-containing chimeric proteins should extensively expand the applications of the streptavidin-biotin system, and offer a variety of applications as new biological tools.

    Title Implications of the Human Genome Project for Clinical Neurosurgery.
    Date May 1991
    Journal Clinical Neurosurgery
    Title Structural and Transcriptional Analysis of a Human Subtelomeric Repeat.
    Date May 1991
    Journal Nucleic Acids Research
    Excerpt

    A human subtelomeric repeat (designated as the HST repeat) has been isolated and characterized from a yeast artificial chromosome containing one human telomere. This repeat is located immediately adjacent to the telomeric T2AG3 repeats at the extreme termini of the human chromosomes. The DNA sequence of 3.6 kb of the HST repeat has been determined. The HST repeat spans over 3.6 kb in length, and contains one evolutionarily conserved CpG-rich region. The copy number of the HST repeat varies among telomeres. Genomic hybridization experiments suggest that the HST repeat consists of two distinct segments, and the distal portions of the HST repeat are also distributed elsewhere in the genome. In HeLa cells, the HST repeat sequence appears to be transcribed into a 6 kb polyadenylated RNA and a variety of non-polyadenylated RNA species.

    Title New Techniques for Physical Mapping of the Human Genome.
    Date March 1991
    Journal The Faseb Journal : Official Publication of the Federation of American Societies for Experimental Biology
    Excerpt

    We describe improvements in techniques and strategies used for making maps of the human genome. The methods currently used are changing and evolving rapidly. Today's techniques can produce ordered arrays of DNA fragments and overlapping sets of DNA clones covering extensive genomic regions, but they are relatively slow and tedious. Methods under development will speed the process considerably. New developments include a range of applications of the polymerase chain reaction, enhanced procedures for high resolution in situ hybridization, and improved methods for generating, manipulating, and cloning large DNA fragments. More detailed genetic and physical maps will be useful for finding genes, including those associated with human diseases, long before the complete DNA sequence of the human genome is available.

    Title Cerebral Vasculopathy and Infarction in a Woman with Carcinomatous Meningitis.
    Date January 1991
    Journal Journal of Neuro-oncology
    Title Cd4-t-cell Antigen Receptor Complexes on Human Leukemia T Cells.
    Date August 1990
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    CD4 and T-cell antigen receptor (TCR) comodulate from the surface of human and murine T cells following exposure to monoclonal anti-CD4 or anti-TCR. This comodulation may occur because expression of CD4 and TCR is regulated by similar transmembrane signals or because CD4 and TCR are physically associated. To study multimolecular assemblies on the plasma membrane, we developed a flow cytometric method for detecting singlet-singlet energy transfer between fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated monoclonal antibodies as sensitized TRITC emission on intact, single cells. Using this procedure, we detected CD4-TCR complexes on the surface of the transformed human leukemia T cells, HPB-ALL, in the absence of stimulation. More than one CD4 were found in association with one TCR. CD4-TCR complexes were not in rapid equilibrium with free CD4 and free TCR, and they were not induced by the dye-labeled anti-CD4 or anti-TCR.

    Title Nucleosome Assembly of Simian Virus 40 Dna in a Mammalian Cell Extract.
    Date June 1990
    Journal Molecular and Cellular Biology
    Excerpt

    We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.

    Title Hugo Physical Mapping.
    Date June 1990
    Journal Nature
    Title Evolving Strategies for Making Physical Maps of Mammalian Chromosomes.
    Date May 1990
    Journal Genome / National Research Council Canada = Génome / Conseil National De Recherches Canada
    Excerpt

    Two types of physical maps are described: restriction maps made by top down approaches using enzymes that cut the genome infrequently, and complete libraries, made by bottom up approaches using fingerprinting of randomly selected cloned DNA. Construction of such maps for mammalian chromosomes is complicated by the mosaic nature of mammalian genomes, and extensive polymorphisms at the cleavage sites of most enzymes that yield large DNA fragments. However, it appears that both of these potential difficulties can be turned into advantages by new mapping strategies. When combined with yeast artificial chromosome cloning and polymerase chain reaction amplification methods, these approaches should soon yield complete maps of many human chromosomes.

    Title Orchestrating the Human Genome Project.
    Date May 1990
    Journal Science (new York, N.y.)
    Excerpt

    The Human Genome Project is under way. The Department of Energy and the National Institutes of Health are cooperating effectively to develop organizational structures and scientific priorities that should keep the project on schedule and within its budget.

    Title Cooperative Biotin Binding by Streptavidin. Electrophoretic Behavior and Subunit Association of Streptavidin in the Presence of 6 M Urea.
    Date March 1990
    Journal The Journal of Biological Chemistry
    Excerpt

    We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.

    Title Expression of a Cloned Streptavidin Gene in Escherichia Coli.
    Date February 1990
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We describe the construction of systems for expressing the cloned streptavidin gene in Escherichia coli. Although the streptavidin gene is extremely lethal to the host cells, because of the strong biotin binding of the gene product, the gene was expressed efficiently in E. coli by using T7 RNA polymerase/T7 promoter expression systems. The expressed streptavidin accumulated to more than 35% of the total cell protein. The expressed streptavidin was insoluble in the cell. However, after solubilization by dialysis against 6 M guanidine hydrochloride (pH 1.5) and removal of guanidine hydrochloride by dialysis, the protein became soluble and renatured. This simple procedure yielded streptavidin purified almost to homogeneity. The purified streptavidin bound 3.5-3.9 molecules of biotin per molecule, indicating that it had almost full biotin-binding ability. Some of the purified streptavidin molecules aggregated into oligomers, suggesting that the C-terminal region of the molecule, present in our material but absent in typical preparations, may be responsible for the aggregation.

    Title The Huntington Disease Locus is Most Likely Within 325 Kilobases of the Chromosome 4p Telomere.
    Date January 1990
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The genetic defect responsible for Huntington disease was originally localized near the tip of the short arm of chromosome 4 by genetic linkage to the locus D4S10. Several markers closer to Huntington disease have since been isolated, but these all appear to be proximal to the defect. A physical map that extends from the most distal of these loci, D4S90, to the telomere of chromosome 4 was constructed. This map identifies at least two CpG islands as markers for Huntington disease candidate genes and places the most likely location of the Huntington disease defect remarkably close (within 325 kilobases) to the telomere.

    Title Isolation and Characterization of a Human Telomere.
    Date September 1989
    Journal Nucleic Acids Research
    Excerpt

    A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.

    Title Use of Fluorescent Probes to Study Nucleosomes.
    Date September 1989
    Journal Methods in Enzymology
    Title Recycling Class I Mhc Antigens: Dynamics of Internalization, Acidification, and Ligand-degradation in Murine T Lymphoblasts.
    Date August 1989
    Journal The Journal of Molecular and Cellular Immunology : Jmci
    Excerpt

    We have previously shown that activated T lymphocytes spontaneously internalize their own surface class I MHC antigens and that this phenomenon is specific for these cells since it does not occur in B lymphocytes even after activation. The present work was aimed at defining the quantitative aspects of this phenomenon and, in particular, at the elucidation of the route of the internalized class I MHC antigens. We intended to determine if the internalized molecules are delivered to the lysosomal compartment and digested there or if instead they are brought back to the plasma membrane in a recycling pathway similar to that described for various cell surface proteins known to be engaged in the process of receptor-mediated endocytosis. We have devised a flow cytometric assay based on the use of fluorochrome-labeled monoclonal anti-H-2K antibodies to measure the kinetics of H-2K internalization. Comparison of the time necessary for the internalization of one-half of the surface H-2K molecules in activated T lymphocytes, which is approximately 1 hour, with the half-life of these molecules on the same cells, which is 14 hours, clearly indicates that the internalized molecules are not degraded but are instead recycled. The recycling takes place in an endosomal compartment with an average pH of about 5.6. The monoclonal anti-H-2K antibody used in these studies was not eluted from the H-2K molecules at this pH and is recycled along with them. On the other hand, protein A bound to the Fc of the anti-H-2K antibody was eluted at the low pH of the endosomes, delivered to lysosomes, and digested. We have therefore defined a novel phenomenon, namely the recycling of class I MHC antigens, which occurs selectively in T lymphocytes. The features of this phenomenon are similar to the recycling of surface receptors which mediate the endocytosis of a variety of extracellular ligands in different cells. However, no physiological extracellular ligand is known for class I MHC antigens. It is a reasonable speculation that upon activation T lymphocytes recycle their own surface class I MHC antigens as part of the complex machinery whereby these lymphocytes recognize and respond to non-self moieties on the plasma membranes of presentor or target cells.

    Title Construction of a Not I Restriction Map of the Fission Yeast Schizosaccharomyces Pombe Genome.
    Date June 1989
    Journal Nucleic Acids Research
    Excerpt

    Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.

    Title High-resolution Separation and Accurate Size Determination in Pulsed-field Gel Electrophoresis of Dna. 3. Effect of Electrical Field Shape.
    Date May 1989
    Journal Biochemistry
    Excerpt

    The resolution of pulsed-field gel electrophoresis is dramatically affected by the number and configuration of the electrodes used, because these alter the shape of the applied electrical fields. Here we present calculations and experiments on the effect of electrode position in one of the most commonly used pulsed-field gel electrophoresis configurations. The goal was to explore which aspects of the electrical field shape correlate with improved electrophoretic resolution. The most critical variable appears to be the angle between the alternate electrical fields. The most effective electrode configurations yield angles of more than 110 degrees. A continually increasing angle between the fields produces band sharpening that greatly enhances the resolution.

    Title High-resolution Separation and Accurate Size Determination in Pulsed-field Gel Electrophoresis of Dna. 4. Influence of Dna Topology.
    Date May 1989
    Journal Biochemistry
    Excerpt

    Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.

    Title High-resolution Separation and Accurate Size Determination in Pulsed-field Gel Electrophoresis of Dna. 1. Dna Size Standards and the Effect of Agarose and Temperature.
    Date May 1989
    Journal Biochemistry
    Excerpt

    Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.

    Title High-resolution Separation and Accurate Size Determination in Pulsed-field Gel Electrophoresis of Dna. 2. Effect of Pulse Time and Electric Field Strength and Implications for Models of the Separation Process.
    Date May 1989
    Journal Biochemistry
    Excerpt

    Bacteriophage DNAs annealed into linear oligomeric concatemers were used to examine the quantitative pulsed-field gel electrophoretic behavior of different-sized DNAs as a function of electrical field strength and pulse time. Three zones of resolution are observed for increasingly larger DNAs. In the first two zones, the electrophoretic mobility decreases linearly with increasing DNA size. The separation in zone 2 is roughly twice that in zone 1. The largest DNA molecules do not resolve at all and migrate in a compression zone. Mobility in zone 1 increases linearly with the electric field strength and decreases with the inverse of the pulse time. The behavior of DNA in zone 2 is qualitatively similar. However, the effect of field strength and pulse time on the separations in each zone is quite different. The results for zone 1 are generally consistent with the predictions of several existing physical models of pulsed-field gel electrophoresis, but no model accounts for all of the observed behavior in the three zones.

    Title Mapping the Genome.
    Date March 1989
    Journal Basic Life Sciences
    Title Synthesis and Properties of Novel Psoralen Derivatives.
    Date January 1989
    Journal Biochemistry
    Excerpt

    We have synthesized a set of new trimethylpsoralen derivatives that are characterized by a chain extending from the 4'-position of the furan ring and linked to this ring by an aminomethylene group. The nature of the side chain can be varied widely. In these derivatives, the chains contain either amino or ethylene oxide units for enhanced water solubility and allow the introduction of a thiol or amine group to nucleic acids. These compounds represent the first set of thiolated psoralen derivatives, and their usefulness is demonstrated in several nucleic acid cross-linking experiments. The reagents can be used to create both intraduplex reversible cross-links between the two single-strand partners in a DNA double helix and interduplex reversible cross-links between two DNA double helices.

    Title Preparation and Characterization of Biotinylated Psoralen.
    Date October 1988
    Journal Nucleic Acids Research
    Excerpt

    Biotinylated psoralen (BPsor), a psoralen derivative containing a biotin moiety attached via a long-chain, positively charged linker, has been synthesized and its interactions with DNA and avidin have been studied. As do other psoralen derivatives, BPsor photoreacts with DNA to form interstrand crosslinks. The biotin binds to streptavidin after the reaction of BPsor with DNA, and this property has been used to measure low levels of BPsor modified DNA by ELISA with streptavidin and biotinylated alkaline phosphatase. In addition, BPsor retains the biological activity of psoralen, as shown by its ability to inhibit lymphocyte proliferation at a level of 10 ng/ml.

    Title Pulsed-field Gel Electrophoresis of Very Large Dna Molecules.
    Date September 1988
    Journal Annual Review of Biophysics and Biophysical Chemistry
    Title Content and Organization of the Human Ig Vh Locus: Definition of Three New Vh Families and Linkage to the Ig Ch Locus.
    Date September 1988
    Journal The Embo Journal
    Excerpt

    We present a detailed analysis of the content and organization of the human immunoglobulin VH locus. Human VH genes representing five distinct families were isolated, including novel members belonging to two out of three of the known VH gene families (VH1 and VH3) as well as members of three new families (VH4, VH5, and VH6). We report the nucleotide sequence of 21 novel human VH genes, many of which belong to the three new VH gene families. In addition, we provide a preliminary analysis of the organization of these gene segments over the full extent of the locus. We find that the five multi-segment families (VH1-5) have members interspersed over nearly the full 1500-2000 kb of the VH locus, and estimate that the entire heavy chain locus covers 2500 kb or less. Finally, we provide the first report of the physical linkage of the variable and constant loci of a human Ig gene family by demonstrating that the most proximal known human VH segments lie within 100 kb of the constant region locus.

    Title A Stable Complex Between Homopyrimidine Oligomers and the Homologous Regions of Duplex Dnas.
    Date May 1988
    Journal Nucleic Acids Research
    Excerpt

    When plasmid DNA duplexes carrying the regular homopurine-homopyrimidine inserts (dGA)n, (dTC)n and (dG)n, (dC)n are preincubated with homologous labeled oligo(dPy) ((dTC)n and (dC)n respectively) at acid pH, the label co-electrophoreses with the duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH greater than 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed complex appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form.

    Title Strategies for Mapping and Cloning Macroregions of Mammalian Genomes.
    Date March 1988
    Journal Methods in Enzymology
    Title Purification, Specific Fragmentation, and Separation of Large Dna Molecules.
    Date March 1988
    Journal Methods in Enzymology
    Title Studies on the Arrangement of Dna Inside Viruses Using a Breakable Bis-psoralen Crosslinker.
    Date March 1988
    Journal Journal of Molecular Biology
    Excerpt

    We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40. Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments. In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model. These results imply that the structures of DNA inside these viruses are either highly disordered or very complex.

    Title Characterization and Crystallization of Core Streptavidin.
    Date November 1987
    Journal The Journal of Biological Chemistry
    Excerpt

    We have characterized a streptavidin product that had been reduced to a minimal size that still retained full biotin-binding activity. This core streptavidin is proteolyzed at both ends at points that correspond closely with the termini of hen egg white avidin. Core streptavidin is more soluble than is the parent molecule. We have grown three different types of crystals of core streptavidin. The symmetry properties of these crystals prove that the molecule is a tetramer organized in tetrahedral (D2) point symmetry. The crystallographic response to the interaction of biotin with core streptavidin indicates that some conformational change accompanies ligand binding. We are attempting to determine the three-dimensional structure of streptavidin and its complex with selenobiotin from these crystals of core streptavidin.

    Title Long-range and Molecular Mapping of the Human Major Histocompatibility Complex.
    Date August 1987
    Journal Transactions of the Association of American Physicians
    Title Sequences That Adopt Non-b-dna Conformation in Form V Dna As Probed by Enzymic Methylation.
    Date July 1987
    Journal Journal of Molecular Biology
    Excerpt

    pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero. We have used sequence-specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA. Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation. Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences. Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure. In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA. These non-B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain. Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed. In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.

    Title An Electrophoretic Karyotype for Schizosaccharomyces Pombe by Pulsed Field Gel Electrophoresis.
    Date July 1987
    Journal Nucleic Acids Research
    Excerpt

    The three chromosomal DNAs of S. pombe have been fractionated by pulsed field gel electrophoresis. The resulting molecular karyotype will greatly speed gene mapping in this organism, and it indicates that the separation range of the technique extends to DNA molecules as large as 9,000,000 base pairs.

    Title A Physical Map of the Escherichia Coli K12 Genome.
    Date July 1987
    Journal Science (new York, N.y.)
    Excerpt

    A physical map of a genome is the structure of its DNA. Construction of such a map is a first step in the complete characterization of that DNA. The restriction endonuclease Not I cuts the genome of Escherichia coli K12 into 22 DNA fragments ranging from 20 kilobases (20,000 base pairs) to 1000 kilobases. These can be separated by pulsed field gel electrophoresis. The order of the fragments in the genome was determined from available E. coli genetic information and analysis of partial digest patterns. The resulting ordered set of fragments is a macrorestriction map. This map facilitates genetic and molecular studies on E. coli, and its construction serves as a model for further endeavors on larger genomes.

    Title Approaches to Physical Mapping of the Human Genome.
    Date June 1987
    Journal Cold Spring Harbor Symposia on Quantitative Biology
    Title Molecular Approaches to the Characterization of Megabase Regions of Dna: Applications to the Human Major Histocompatibility Complex.
    Date June 1987
    Journal Cold Spring Harbor Symposia on Quantitative Biology
    Title Megabase-scale Mapping of the Hla Gene Complex by Pulsed Field Gel Electrophoresis.
    Date April 1987
    Journal Science (new York, N.y.)
    Excerpt

    In the study of the genetic structure of mammalian chromosomes, there exists a "resolution gap" between molecular cloning experiments and meiotic linkage analyses. This gap has discouraged attempts to construct full-scale genetic maps of mammalian chromosomes. The organization of the human major histocompatibility complex was examined within this range by pulsed field gel electrophoresis. The data obtained indicate that the complex spans over 3000 kilobases and enable the construction of a megabase-scale molecular map. These results indicate that the techniques employed in DNA extraction, enzymatic digestion, electrophoresis, and hybridization are suitable for the efficient analysis of megabase regions of mammalian chromosomes and effectively bridge the resolution gap between molecular cloning and classical genetics.

    Title Actinomycin D Facilitates Transition of At Domains in Molecules of Sequence (at)nagct(at)n to a Dnase I Detectable Alternating Structure.
    Date April 1987
    Journal Nucleic Acids Research
    Excerpt

    The interaction of actinomycin D with (AT)nAGCT(AT)n (where n = 2, 3, or 4) was investigated using a combination of imino proton NMR and DNAse I digestion. The stoichiometry of the interaction appears to be one:one with the actinomycin chromophore intercalated between the two GC base pairs. This binding event facilitates the conversion of the flanking repetitive AT regions to an alternating conformation characterized by induced sensitivity of the ApT sequences to attack by DNAse I. The neighboring TpA sequences do not exhibit rate changes as a function of binding of the drug. The potential relevance of such ligand induced DNA structural alterations is discussed.

    Title Intracellular Accumulation of T-cell Receptor Complex Molecules in a Human T-cell Line.
    Date November 1986
    Journal Science (new York, N.y.)
    Excerpt

    This work was aimed at understanding the mechanisms of T-lymphocyte function by studying the cellular distribution and traffic of molecules of the T-cell receptor complex. The accumulation of specific molecules in intracytoplasmic vesicles is related to the activation of T lymphocytes. Some of these molecules include acid hydrolases, the transferrin receptor, and class I antigens of the major histocompatibility complex. Molecules of the T-cell receptor complex have now also been found in intracytoplasmic vesicles in a human T-cell line derived from a lymphoblastic leukemia. Such vesicles were tightly associated with the cytoplasmic microtubule network. One functional aspect of this association is a cellular pathway by which vesicles traveling to and from the cell surface converge in an area of the cells that is rich in processing enzymes.

    Title Studies of Tobacco Mosaic Virus Reassembly with an Rna Tail Blocked by a Hybridised and Cross-linked Probe.
    Date June 1986
    Journal European Journal of Biochemistry / Febs
    Excerpt

    Segments of cloned cDNA to tobacco mosaic virus RNA, 150--300-bases long, have been hybridised and cross-linked to the RNA, which has then been used for reassembly experiments. This enables the elongation reaction, which does not encapsidate the double-stranded region generated, to be stopped at specific regions along the RNA and the resulting particles to be characterised, by measuring the lengths of the rods in the electron microscope. With hybridisation to the 3'-tail the entire RNA contiguous to the nucleation region is encapsidated, from the 5'-terminus up to the modified region. When the double-stranded region is on the 5'-side of the nucleation region, the mean length of the particles corresponds to a situation in which the double-stranded region is unable to enter the central hole of the growing rod, but the 3'-tail of the RNA is completely encapsidated. The longest particles hybridised on the 5'-tail (i.e. in a class longer than the mean length) show an effect complementary to those with a 3'-block, and have lengths which correspond to encapsidation from the modified region to the 3'-terminus, despite the continued presence of the 5'-tail up the rod. In all cases where there is a remaining 5'-tail the lengths observed can only be explained if elongation has occurred substantially, or probably completely, along the 3'-tail. Hence elongation must have occurred simultaneously along both the 5' and 3'-tails of the tobacco mosaic virus RNA after initiation on the internal nucleation region.

    Title Effect of Trypsinization and Histone H5 Addition on Dna Twist and Topology in Reconstituted Minichromosomes.
    Date June 1986
    Journal Nucleic Acids Research
    Excerpt

    Free DNA in solution exhibits an untwisting of the double helix with increasing temperature. We have shown previously that when DNA is reconstituted with histones to form nucleosome core particles, both the core DNA and the adjacent linker DNA are constrained from thermal untwisting. The origin of this constraint is unknown. Here we examine the effect of two modifications of nucleosome structure on the constraint against thermal untwisting, and also on DNA topology. In one experiment, we removed the highly positively charged histone amino and carboxy termini by trypsinization. Alternatively, we added histone H5, a histone H1 variant from chick erythrocytes. Neither of these modifications had any major effect on DNA topology or twist in the nucleosome.

    Title Karyotype Analysis of Leishmania Species and Its Use in Classification and Clinical Diagnosis.
    Date May 1986
    Journal Science (new York, N.y.)
    Excerpt

    Chromosomes of four species of Leishmania represented by ten different geographic isolates were analyzed by pulsed field gradient gel electrophoresis (PFG) to assess chromosome stability in these parasitic protozoans. Among different geographic isolates of the same subspecies, more than two-thirds of chromosomes had similar sizes, ethidium bromide staining intensities, and locations of alpha,beta-tubulin genes. However, among New World Leishmania, members of different species or subspecies have fewer than one-third of their chromosomes in common. Therefore, PFG karyotypes of Leishmania exhibit intraspecific variability similar to that reported for other parasitic protozoans. The greater similarities of the karyotypes of members of the same Leishmania subspecies may indicate that they represent valid taxa. These similarities also allowed the use of PFG in clinical diagnosis for rapid and accurate typing of patient isolates.

    Title Molecular Cloning and Nucleotide Sequence of the Streptavidin Gene.
    Date April 1986
    Journal Nucleic Acids Research
    Excerpt

    Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region and the 3' and 5' flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure in both proteins and considerable overall structural similarity between them.

    Title Nucleosomes Are Phased Along the Mouse Beta-major Globin Gene in Erythroid and Nonerythroid Cells.
    Date April 1986
    Journal Cell
    Excerpt

    We have used the chemical cleavage reagent methidiumpropyl-EDTA-Fe(II) to determine the location of the nucleosomes along the mouse beta-major globin gene in erythroid and nonerythroid cells. In mouse L cells, in which the globin gene is inactive, the nucleosomes are precisely positioned with respect to the underlying DNA sequence from positions -3000 to +1500 relative to the cap site. In uninduced and induced murine erythroleukemia cells, the same phasing persists but is interrupted from positions -200 to +500. This gap in the phased distribution of nucleosomes appears to be protected from MPE-Fe(II) digestion, and is bounded on both sides by hypersensitive sites. These results define at least two structural states for the globin gene: an inactive state in which the gene is covered with a continuous array of phased nucleosomes and an active state in which this array is disrupted over the 5' half of the structural gene.

    Title Purification of Mbo Ii Methylase (gaagma) from Moraxella Bovis: Site Specific Cleavage of Dna at Nine and Ten Base Pair Sequences.
    Date December 1985
    Journal Nucleic Acids Research
    Excerpt

    The restriction modification methylase M. Mbo II has been purified using a sensitive oligonucleotide linker assay. The enzyme methylates the Mbo II recognition sequence* GAAGA at adenine to produce GAAGmA. M. Mbo II can be used in conjunction with the methylation dependent restriction endonuclease Dpn I (GmATC) to produce cleavage at the 10 base sequence GAAGATCTTC. When M. Mbo II is used in combination with M. Cla I (ATCGATCGAT), cleavage by Dpn I occurs at the four ten base sequences GAAGATCTTC, GAAGATCGAT, ATCGATCTTC and ATCGATCGAT, which is equivalent to a nine base recognition site. The use of combinations of adenine methylases and Dpn I to generate highly selective DNA cleavages at a variety of sequences up to fourteen base pairs is discussed.

    Title Structure of the Escherichia Coli 16 S Ribosomal Rna. Psoralen Crosslinks and N-acetyl-n'-(p-glyoxylylbenzoyl)cystamine Crosslinks Detected by Electron Microscopy.
    Date October 1985
    Journal Journal of Molecular Biology
    Excerpt

    Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.

    Title Nucleosome Core Particles Suppress the Thermal Untwisting of Core Dna and Adjacent Linker Dna.
    Date August 1985
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Covalently closed circular DNA is known to undergo a temperature-dependent change in helical twist. We have reconstituted nucleosome core particles onto closed circular DNA and measured the thermal untwisting of the DNA as a function of nucleosome density. The results demonstrate that the DNA associated with the nucleosome core particle does not alter its twist when the temperature is varied between 4 degrees C and 37 degrees C, and that the length of DNA prevented from thermal untwisting includes the linker as well as the core DNA.

    Title Analysis of Rna Structure by Ultraviolet Crosslinking and Denaturation Gel Electrophoresis.
    Date August 1985
    Journal Analytical Biochemistry
    Excerpt

    Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species. Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms. The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits. Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts.

    Title Heat Shock Genes: Regulatory Role for Differentiation in Parasitic Protozoa.
    Date July 1985
    Journal Science (new York, N.y.)
    Excerpt

    The parasitic protozoa Trypanosoma brucei and Leishmania major are transmitted by insect vectors to their mammalian hosts. The temperature difference between the hosts (25 degrees and 37 degrees C) may induce a heat shock response in the parasite. Transcripts of heat shock genes (homologous to Hsp70 and Hsp83) were 25 to 100 times more abundant in Trypanosoma brucei bloodstream forms (trypomastigotes) than in insect (procyclic) stages. In Leishmania major the patterns of heat shock gene expression in promastigotes (insect-adapted) and amastigotes (mammal-adapted) were different. A temperature shift in vitro induced differentiation of Leishmania major from promastigotes to amastigotes. Therefore, heat shock genes may be responsible for differentiation of these vector-borne parasites.

    Title Torsional Properties of Dna in Chromatin.
    Date June 1985
    Journal Progress in Clinical and Biological Research
    Title Structure of Ribosome-bound Messenger Rna As Revealed by Enzymatic Accessibility Studies.
    Date May 1985
    Journal Journal of Molecular Biology
    Excerpt

    Digestion with ribonuclease T2 has been used to study the size of poly(U) protected by ribosome binding. Several different preparations of ribosomes all appear to cover 49 nucleotides of message; however, there are two partially accessible internal nuclease cleavage sites, which yield, ultimately, fragments 20, 16 and 13 nucleotides in length. Curiously, the site between fragments of length 20 and 16 is accessible to RNase T2 but not to the several much smaller RNases. Arguments based on the quantitative pattern of cleavage and comparisons with previous studies lead to the conclusion that the 20-mer is the 5' fragment, while 13-mer (which is lost the moment it is cleaved from the 16-mer) is the 3' fragment. Both ribosome-bound tRNAs appear to contact only the 16-mer. The presence of the two internal cleavage sites fits nicely with recent electron microscopic data suggesting that mRNA forms a loop around the 30 S subunit.

    Title Enzymatic Synthesis of Uniformly 32p-labeled Polyribonucleotides and High-specific-activity Ribonucleoside 5'-[alpha-32p]diphosphates.
    Date May 1985
    Journal Analytical Biochemistry
    Excerpt

    Uniformly 32P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5'-[alpha-32P]triphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition.

    Title Mapping the Location of Psoralen Crosslinks on Rna by Mung Bean Nuclease Sensitivity of Rna.dna Hybrids.
    Date April 1985
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    An indirect high resolution method has been developed for finding the location of intrastrand crosslinks in RNA. An end-labeled DNA strand that overlaps the approximate crosslink position is hybridized to the RNA and then treated with mung bean nuclease. The resulting digest is analyzed on a sequencing-type gel. The method was tested with the major psoralen crosslink seen in the 16S rRNA of inactivated Escherichia coli 30S ribosomal subunits. This crosslink was previously mapped between residues 930 +/- 25 and a region close to the 3' end by electron microscopy. The new indirect method reveals that the crosslink occurs between residues 919 and 923 and residues 1530 and 1534. When these results are examined in the light of existing consensus secondary structure models for the 16S rRNA, it appears that the Shine-Dalgarno sequence is located close to the peptidyl tRNA binding site.

    Title The Complete Pattern of Mutagenesis Arising from the Repair of Site-specific Psoralen Crosslinks: Analysis by Oligonucleotide Hybridization.
    Date February 1985
    Journal Nucleic Acids Research
    Excerpt

    Psoralen crosslinks were site-specifically placed in plasmid pBR322 near the BamHI site in the tet gene by enzymatically inserting mercurated nucleotides and reacting at the target site with a sulfhydryl-containing psoralen. The damaged plasmid was repaired in SOS-induced E. coli cells. Mutants were detected by colony hybridization to oligonucleotides in the target region, and their sequences were determined. The mutations are all base substitutions, 80% transitions and 20% transversions, similar to the mutations previously identified by the loss of tetracycline resistance. However, the mutation sites detected by a physical method, unconstrained by phenotypic changes, follow a broader distribution than those identified genetically. They occur primarily at favored psoralen crosslinking sites, where T-T and T-C interstrand crosslinks can be formed. A majority of these mutations are silent.

    Title Possible Structures of Homopurine-homopyrimidine S1-hypersensitive Sites.
    Date January 1985
    Journal Nucleic Acids Research
    Excerpt

    S1 nuclease hypersensitive sites in the 5' flanking regions of eukaryotic genes, present in small artificial supercoiled DNA circles, reside in homopurine-homopyrimidine stretches. The hierarchical behavior which these sites exhibit is consistent with the notion that they act as sinks of torsional free energy. By employing DMS as a single-strand-specific reagent, we show that these sites (despite their S1 sensitivity) are regions of duplex DNA. A simple thermodynamic treatment indicates that the high torsional stress in the small DNA circles is almost certain to be relieved by the formation of alternate DNA structures. The same treatment places some constraints on the types and sizes of the regions with alternate conformation. While no definitive structural conclusions can be drawn, left-handed helices seem most consistent with the extent and the pattern of sensitivity to S1 nuclease.

    Title Internalization and Acidification of Insulin by Activated Human Lymphocytes.
    Date November 1984
    Journal Journal of Cellular Physiology
    Excerpt

    The binding and internalization of fluorescein isothiocyanate-conjugated insulin by nonactivated and phytohemagglutinin-activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor-mediated endocytosis and acidified within 60 min after addition of insulin. Dual-fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.

    Title Mutagenic Sos Repair of Site-specific Psoralen Damage in Plasmid Pbr322.
    Date November 1984
    Journal Journal of Molecular Biology
    Excerpt

    The mutagenic repair of psoralen damage was examined by transforming Escherichia coli with psoralen-treated pBR322. Plasmid DNA randomly reacted with psoralen was repaired only when the E. coli was uvrA+ and recA+, and only when the cells were pre-irradiated with far-ultraviolet light. The recA dependence and requirement for pre-irradiation are characteristics of SOS repair. Psoralens were placed specifically near the BamHI site, in the tetracycline-resistance gene of pBR322, using a sulfhydryl-containing psoralen derivative. Repair of this damage also required pre-irradiation of the host cells. This repair was accompanied by a 4% frequency of mutagenesis to a tetracycline-sensitive phenotype. Sequence analysis of these mutant plasmids revealed that 75% had mutations within the targeted region, while 25% had no sequence changes within 100 bases of the BamHI site. In up to five independent isolates only one kind of mutation was observed at each site, suggesting that mutagenic SOS repair is influenced by DNA structure at the site of the psoralen. Most mutations were transitions, primarily G-C to A-T changes. Some transitions occurred at sites where psoralen crosslinks could not have formed, and these may have arisen from the repair of psoralen monoadducts.

    Title Acidification of Internalized Class I Major Histocompatibility Complex Antigen by T Lymphoblasts.
    Date November 1984
    Journal Cellular Immunology
    Excerpt

    It has previously been shown that activated murine T lymphocytes express intracellular vesicles containing the class I major histocompatibility complex (MHC) antigen H-2K. Evidence has also been provided that such vesicles may be part of a cellular pathway of spontaneous H-2K antigen internalization and recycling, which is specific to T-lymphoid cells. Dual fluorescence flow cytometry has now been used to establish that H-2K antigen is acidified upon internalization in concanavalin A-stimulated but not lipopolysaccharide-stimulated murine splenocytes, thus providing further support that in T lymphoblasts this class I MHC antigen may travel intracellular routes similar to those reported for other cell surface receptors.

    Title Wavelength Dependence for Amt Crosslinking of Pbr322 Dna.
    Date November 1984
    Journal Photochemistry and Photobiology
    Title Reduced Insulin Endocytosis in Serum-transformed Fibroblasts Demonstrated by Flow Cytometry.
    Date July 1984
    Journal Cytometry
    Excerpt

    Neoplastic transformation often results in the loss of growth control and concomitant changes in cell surface properties. The changes in endocytosis of a variety of probes after serum or anchorage transformation were measured for mouse fibroblasts by flow cytofluorometry. No major differences in dextran (fluid phase) or histone (nonspecific-adsorptive) endocytosis were observed among four cell lines with different growth properties. However, decreased receptor-mediated internalization of alpha 2-macroglobulin was observed for cell lines transformed to either serum or anchorage independence. Furthermore, increased wheat germ agglutinin and decreased insulin endocytosis were observed, but only in serum transformants. The changes specific to serum transformants were not accounted for by changes in binding of wheat germ agglutinin or insulin. The possible implications of these observations regarding serum transformation and the insulin requirement for growth in serum-free medium are discussed.

    Title Antigenic Variation in Trypanosoma Brucei Analyzed by Electrophoretic Separation of Chromosome-sized Dna Molecules.
    Date June 1984
    Journal Cell
    Excerpt

    Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.

    Title Separation of Yeast Chromosome-sized Dnas by Pulsed Field Gradient Gel Electrophoresis.
    Date June 1984
    Journal Cell
    Excerpt

    A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.

    Title Endosome Ph Measured in Single Cells by Dual Fluorescence Flow Cytometry: Rapid Acidification of Insulin to Ph 6.
    Date June 1984
    Journal The Journal of Cell Biology
    Excerpt

    The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.

    Title Rna Splicing in Neurospora Mitochondria: Structure of the Unspliced 35s Precursor Ribosomal Rna Detected by Psoralen Cross-linking.
    Date April 1983
    Journal Cell
    Excerpt

    The structure of the unspliced 35S precursor rRNA of Neurospora mitochondria was studied by psoralen photochemical cross-linking. The results show that when the 35S RNA is cross-linked in ribonucleoprotein particles (RNPs) under appropriate conditions, the predominant configuration is a 2.2 kb intron loop which brings opposite splice sites into proximity; that the predominant secondary structural feature in the free RNA is a relatively large hairpin (length = 0.105 kb) in the center of the molecule at or near the 5' splice site; that the intron loop and the central hairpin are different configurations of sequences at or near the 5' splice site; and that the intron loop is stabilized by protein components of RNPs. Based on the structures detected by psoralen photochemical cross-linking, we propose a mechanism for the splicing of the Neurospora mitochondrial precursor rRNA. We propose further that certain features of this mechanism may be relevant to the splicing of other RNAs, including eucaryotic mRNAs.

    Title Conserved 5s Rrna Complement to Trna is Not Required for Protein Synthesis.
    Date March 1982
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The notion that tRNA and 5S rRNA interact through evolutionarily conserved complementary sequences has been tested by nucleolytic modification of the 5S rRNA, using the modified rRNA to reconstitute the large ribosomal subunit, and assaying for poly(uridylic acid)-directed polyphenylalanine synthesis. The 5S rRNA sequence C-G-A-A (residues 43-46) and several residues surrounding it are not essential for protein synthesis.

    Title Structure and Topology of 16s Ribosomal Rna. An Analysis of the Pattern of Psoralen Crosslinking.
    Date January 1981
    Journal Nucleic Acids Research
    Excerpt

    All of the eleven psoralen crosslinking features previously mapped by electron microscopy of the 16S rRNA can be matched by energetically stable duplex regions found by a computer search of the sequence. One additional particularly stable duplex found in the sequence has subsequently been seen in the microscope. There is no indication that a best or unique fit of the sequence to the crosslinking data has been found. However, it is encouraging that all twelve assignments involve non-overlapping regions of the sequence which allows their simultaneous construction. The resulting general pattern of secondary structure is very different from previous suggestions for rRNAs or other complex RNAs. It is an RNA chain folded in space much like a typical protein chain. Because of the interwound nature of double helices, base pairing between distant regions of the sequence might result in topologically knotted structures. However, examination of available electron microscopic data suggests that the 16S rRNA does not contain any knots.

    Title Base-pairing Between Distant Regions of the Escherichia Coli 16 S Ribosomal Rna in Solution.
    Date April 1980
    Journal Journal of Molecular Biology
    Title Crosslinking Studies on the Organization of the 16 S Ribosomal Rna Within the 30 S Escherichia Coli Ribosomal Subunit.
    Date April 1980
    Journal Journal of Molecular Biology
    Title Salt-induced Structural Changes of Nucleosome Core Particles.
    Date November 1979
    Journal Journal of Molecular Biology
    Title Dynamics of Nucleosome Structure Studied by Fluorescence.
    Date October 1978
    Journal Cold Spring Harbor Symposia on Quantitative Biology
    Title Pyrene Derivatives As Fluorescent Probes of Conformation Near the 3' Termini of Polyribonucleotides.
    Date December 1977
    Journal Biopolymers
    Title Some Features of the Vinblastine-induced Assembly of Porcine Tubulin.
    Date January 1976
    Journal Archives of Biochemistry and Biophysics
    Title The Formation of Filamentous Structures from Iodinated Neurotubules.
    Date November 1975
    Journal Journal of Supramolecular Structure
    Excerpt

    The porcine neurotubule and its basic subunit were found to be modified in vitro by iodination of amino acids (principally tyrosine) using lactoperoxidase. Iodide ion, H2O2, or lactoperoxidase singly or in any pairwise combination had virtually no effect on neurotubules. However, when all three reagents were present, permitting covalent iodination, it was found that at 0.1 iodotyrosines per tubulin dimer the microtubules unravel to form structures which morphologically resemble strands of protofilaments twisted or wound around each other. These abnormal tubules are stable at room temperature and 4 degrees C. Both monomers of tubulin are labeled to approximately the same extent. Iodinated tubulin (0.1 iodotyrosines/dimer) is unable to assemble in vitro under normal assembly conditions. Heavily iodinated microtubules (8 iodines per tubulin dimer) are similar in morphology to the slightly iodinated structures.

    Title Biochemical Studies on the in Vitro Assembly and Disassembly of Microtubules.
    Date October 1975
    Journal Annals of the New York Academy of Sciences
    Title Turbidimetric Studies of the in Vitro Assembly and Disassembly of Porcine Neurotubules.
    Date May 1975
    Journal Journal of Molecular Biology
    Title Investigation of the Ribosomal Peptidyl Transferase Center Using a Photoaffinity Label.
    Date January 1975
    Journal Journal of Molecular Biology
    Title A Dynein-like Protein Associated with Neurotubules.
    Date November 1974
    Journal Febs Letters
    Title Microtubule Assembly in the Absence of Added Nucleotides.
    Date September 1973
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Microtubule assembly is enhanced by the addition of 1 M sucrose or 4 M glycerol to the reassembly mixture. Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions. The tubules assembled in glycerol and sucrose are more stable than tubules formed in the absence of these compounds. Assembly occurs in glycerol or sucrose in the absence of ATP or GTP, but is greatly accelarated by their presence.

    Title Studies on the Hydration of Escherichia Coli Ribosomes by Nuclear Magnetic Resonance.
    Date July 1972
    Journal Journal of Molecular Biology
    Title Modification of Ribonucleic Acid by Chemical Carcinogens. Iv. Circular Dichroism and Proton Magnetic Resonance Studies of Oligonucleotides Modified with N-2-acetylaminofluorene.
    Date April 1972
    Journal Journal of Molecular Biology
    Title Role of Magnesium in the Binding of Tetracycline to Escherichia Coli Ribosomes.
    Date August 1971
    Journal Journal of Molecular Biology
    Title Conformational Studies on Transfer Ribonucleic Acid. Fluorescence Lifetime and Nanosecond Depolarization Measurements on Bound Ethidium Bromidee.
    Date June 1971
    Journal Biochemistry
    Title Coding and Conformational Properties of Oligonucleotides Modified with the Carcinogen N-2-acetylaminofluorene.
    Date October 1970
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The present studies were undertaken to determine the mechanism by which attachment of the carcinogen N-2-acetylaminofluorene to guanosine residues in nucleic acids distors their structure and function. Oligonucleotides were modified with N-acetoxy-2-acetylaminofluorene, repurified, and their base compositions analyzed. Evidence is presented that acetylaminofluorene residues bound to guanosines in GpUpU, ApApG, or poly (U,G) inactivates their function in codon recognition. Circular dichroism spectra suggest that this is caused by gross conformational changes in these compounds involving both a rotation about the glycosidic bond of guanosine residues bearing N-2-acetylaminofluorene, as well as stacking interactions between the drug and bases adjacent to the substituted guanosine.

    Title High Yield Photoreagents for Protein Crosslinking and Affinity Labeling.
    Date
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    4-Nitrophenyl ethers are proposed as new high-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with amines at pH 8 upon irradiation with 366-nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free alcohol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the p-nitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy-4-nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at gamma-cysteine F9 stoichiometrically. Subsequent ultraviolet irradiation yielded a gamma-gamma crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high affinity conformation and that all cooperativity is lost.

    Title Response.
    Date
    Journal Science (new York, N.y.)
    Title Stimulus Dependence of the Flash-lag Effect.
    Date
    Journal Vision Research
    Excerpt

    When two moving objects are presented in perfect alignment, but are not visible for the same amount of time, the briefer object will often be perceived as "lagging" the object of greater duration. Most investigations of this flash-lag effect (FLE) employ high velocity broadband stimuli, such as lines or dots with sharp boundaries and flashes with rapid onset and offset. We introduce a stimulus paradigm with narrow-band stimuli and measure the stimulus dependence of the FLE when basic stimulus parameters of spatio-temporal frequency and temporal duration are varied. We suggest that this dependence is consistent with the involvement of early visual mechanisms and interpret our results in the context of existing theories of the FLE.

    Title Quantitative, Sensitive Analysis of Dna and Rna.
    Date
    Journal Nucleic Acids Symposium Series (2004)
    Excerpt

    DNA or RNA sequences that contain useful information about disease risk, disease occurrence, therapeutic response, and probable prognosis are potentially valuable biomarkers. They can be accessed by biopsy, or in ideal cases non-invasively from easily accessible fluids like blood or urine. Tolls are available for biomarker discovery, validation, and clinical use. Discovery usually requires whole genome analysis, and currently this is done with either nucleic acid arrays (DNA chips) or sequencing. Validation requires high quality data scalability to large numbers of samples. Clinical utility normally needs a high degree of automation, and for non-invasive approaches, great experimental sensitivity and specificity but on small numbers of samples. No one platform can efficiently span the broad range of requirements and project sizes. SEQUENOM uses an automated mass spectrometry platform for the quantitative analysis of DNA and RNA in a variety of settings including genotyping, genecopy number measurements, gene expression, epigenetics, and automated bacterial and viral identification. In collaboration with Amit Meller at Boston University, SEQUENOM is developing optically detected nanospores as a companion platform for its mass spectrometry offering. Both platform use similar homogeneous solution biochemistry to prepare samples. Both platforms depend on vary rapid digital signal processing for real time data analysis and interpretation. The nanopore method uses pores large enough to pass single-stranded DNA but too small to pass double strands. Hence pore passage strips off one of the DNA stands, and optical method are used to detect changes in fluorophores on this strand or attached to reporter probes, as it is stripped. We expect that, when mature, the nanopore method will be extremely const effective for whole genome analysis of genotypes, gene expression, epigenetics, and whole genome sequencing. A key aspect of the nanopore method is its speed. Post sample preparation, many analyses may require only seconds of instrument time.

    Title Obstructive Sleep Apnea in Patients Undergoing Bariatric Surgery-a Tertiary Center Experience.
    Date
    Journal Obesity Surgery
    Excerpt

    The patient population that is evaluated for bariatric surgery is characterized by a very high body mass index (BMI). Since obesity is the most important risk factor for obstructive sleep apnea (OSA), sleep disordered breathing is highly prevalent in this population. If undiagnosed before bariatric surgery, untreated OSA can lead to perioperative and postoperative complications. Debate exists whether all patients that are considered for bariatric surgery should undergo polysomnography (PSG) evaluation and screening for OSA as opposed to only those patients with clinical history or examination concerning sleep disordered breathing. We examined the prevalence and severity of OSA in all patients that were considered for bariatric surgery. We hypothesized that, by utilizing preoperative questionnaires (regarding sleepiness and OSA respiratory symptoms) in combination with menopausal status and BMI data, we would be able to predict which subjects did not have sleep apnea without the use of polysomnography. In addition, we hypothesized that we would be able to predict which subjects had severe OSA (apnea-hypopnea index (AHI) > 30).

    Title Maternal Plasma Dna Sequencing Reveals the Genome-wide Genetic and Mutational Profile of the Fetus.
    Date
    Journal Science Translational Medicine
    Excerpt

    Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to 65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.

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