Biomedical subjects

David C Schwartz

Publications and source records attributed to David C Schwartz.

At least 19 recordsLinked to original sources

PubMed2006

Transchip: single-molecule detection of transcriptional elongation complexes.

A new single-molecule system, Transchip, was developed for analysis of transcription products at their genomic origins. The bacteriophage T7 RNA polymerase and its promoters were used in a model system, and resultant RNAs were imaged and detected at their positions along single template DNA molecules. The Transchip system has drawn from critical aspects of Optical Mapping, a single-molecule system that enables the construction of high-resolution ordered restriction maps of whole genomes from single DNA molecules. Through statistical analysis of hundreds of single-molecule template/transcript complexes, Transchip enables analysis of the locations and strength of promoters, the direction and processivity of transcription reactions, and the termination of transcription. These novel results suggest that the new system may serve as a high-throughput platform to investigate transcriptional events on a large genome-wide scale.

Bacteriophage T7↗

PubMed2006

An algorithm for assembly of ordered restriction maps from single DNA molecules.

The restriction mapping of a massive number of individual DNA molecules by optical mapping enables assembly of physical maps spanning mammalian and plant genomes; however, not through computational means permitting completely de novo assembly. Existing algorithms are not practical for genomes larger than lower eukaryotes due to their high time and space complexity. In many ways, sequence assembly parallels map assembly, so that the overlap-layout-consensus strategy, recently shown effective in assembling very large genomes in feasible time, sheds new light on solving map construction issues associated with single molecule substrates. Accordingly, we report an adaptation of this approach as the formal basis for de novo optical map assembly and demonstrate its computational feasibility for assembly of very large genomes. As such, we discuss assembly results for a series of genomes: human, plant, lower eukaryote and bacterial. Unlike sequence assembly, the optical map assembly problem is actually more complex because restriction maps from single molecules are constructed, manifesting errors stemming from: missing cuts, false cuts, and high variance of estimated fragment sizes; chimeric maps resulting from artifactually merged molecules; and true overlap scores that are "in the noise" or "slightly above the noise." We address these problems, fundamental to many single molecule measurements, by an effective error correction method using global overlap information to eliminate spurious overlaps and chimeric maps that are otherwise difficult to identify.

Algorithms↗

PubMed2006

DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage.

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

Animals↗

PubMed2006

Refinement of optical map assemblies.

MOTIVATION: Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 bp per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bp. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical. RESULTS: We present a computationally efficient model-based method for improving quality of such assemblies. Our method provides very high accuracy even with moderate coverage (<20 x). We utilize a hidden Markov model to represent the consensus map and use the expectation-Maximization algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map. AVAILABILITY: Code is available from www.cmb.usc.edu/people/valouev/

Algorithms↗

PubMed2006

Alignment of optical maps.

We introduce a new scoring method for calculation of alignments of optical maps. Missing cuts, false cuts, and sizing errors present in optical maps are addressed by our alignment score through calculation of corresponding likelihoods. The size error model is derived through the application of Central Limit Theorem and validated by residual plots collected from real data. Missing cuts and false cuts are modeled as Bernoulli and Poisson events, respectively, as suggested by previous studies. Likelihoods are used to derive an alignment score through calculation of likelihood ratios for a certain hypothesis test. This allows us to achieve maximal descriminative power for the alignment score. Our scoring method is naturally embedded within a well known DP framework for finding optimal alignments.

Algorithms↗

PubMed2005

The genome of the kinetoplastid parasite, Leishmania major.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

Animals↗

PubMed2005

High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides.

DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.

Buffers↗

PubMed2005

Whole-genome shotgun optical mapping of Rhodospirillum rubrum.

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.

Contig Mapping↗

PubMed2004

The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism.

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.

Adaptation, Physiological↗

PubMed2004

A microfluidic system for large DNA molecule arrays.

Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.

DNA↗

PubMed2004

An integrative approach for the optical sequencing of single DNA molecules.

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.

DNA-Directed DNA Polymerase↗

PubMed2004

Shear-induced migration in flowing polymer solutions: simulation of long-chain DNA in microchannels [corrected].

We simulate dilute solution dynamics of long flexible polymer molecules in pressure driven flow in channels with widths of roughly 0.1-10 times the polymer bulk radius of gyration. This is done using a self-consistent coarse-grained Langevin description of the polymer dynamics and a numerical simulation of the flow in the confined geometry that is generated by the motions of polymer segments. Results are presented for a model of DNA molecules of approximately 10-100 microm contour length in micron-scale channels. During flow, the chains migrate toward the channel centerline, in agreement with well-known experimental observations. The thickness of the resulting hydrodynamic depletion layer increases with molecular weight at constant flow strength; higher molecular weight chains therefore move with a higher average axial velocity than lower molecular weight chains. In contrast, if the hydrodynamic effects of the confining geometry are neglected, depletion of concentration is observed in the center of the channel rather than at the walls, contradicting experimental observations. The mechanisms for migration are illustrated using a simple kinetic theory dumbbell model of a confined flexible polymer. The simple theory correctly predicts the trends observed in the detailed simulations. We also examine the steady-state stretch of DNA chains as a function of channel width and flow strength. The flow strength needed to stretch a highly confined chain away from its equilibrium length is shown to increase with decreasing channel width, independent of molecular weight; this is fairly well explained using a simple blob picture.

Computer Simulation↗

PubMed2004

Shotgun optical mapping of the entire Leishmania major Friedlin genome.

Leishmania is a group of protozoan parasites which causes a broad spectrum of diseases resulting in widespread human suffering and death, as well as economic loss from the infection of some domestic animals and wildlife. To further understand the fundamental genomic architecture of this parasite, and to accelerate the on-going sequencing project, a whole-genome XbaI restriction map was constructed using the optical mapping system. This map supplemented traditional physical maps that were generated by fingerprinting and hybridization of cosmid and P1 clone libraries. Thirty-six optical map contigs were constructed for the corresponding known 36 chromosomes of the Leishmania major Friedlin genome. The chromosome sizes ranged from 326.9 to 2821.3 kb, with a total genome size of 34.7 Mb; the average XbaI restriction fragment was 25.3 kb, and ranged from 15.7 to 77.8 kb on a per chromosomes basis. Comparison between the optical maps and the in silico maps of sequence drawn from completed, nearly finished, or large sequence contigs showed that optical maps served several useful functions within the path to create finished sequence by: guiding aspects of the sequence assembly, identifying misassemblies, detection of cosmid or PAC clones misplacements to chromosomes, and validation of sequence stemming from varying degrees of finishing. Our results also showed the potential use of optical maps as a means to detect and characterize map segmental duplication within genomes.

Animals↗

PubMed2004

Single-molecule approach to bacterial genomic comparisons via optical mapping.

Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Escherichia coli K12↗

PubMed2003

DNA dynamics in a microchannel.

An extended Brownian dynamics simulation method is used to characterize the dynamics of long DNA molecules flowing in microchannels. The relaxation time increases due to confinement in agreement with scaling predictions. During flow the molecules migrate toward the channel center line, and thereby segregate according to molecular weight. Capturing these effects requires the detailed incorporation of solvent flow in the simulation method, demonstrating the importance of hydrodynamic effects in the dynamics of confined macromolecules.

Computer Simulation↗

PubMed2003

A superfamily of protein tags: ubiquitin, SUMO and related modifiers.

The biological functions of many proteins are altered by their covalent attachment to polypeptide modifiers. The best-known example of this type of modification is ubiquitination. Ubiquitin has a well-documented role in targeting proteins for degradation by the proteasome, but additional effects of protein ubiquitination are now being uncovered. Furthermore, multiple polypeptides that are distinct from, but related to, ubiquitin are also enzymatically coupled to target macromolecules, and these ubiquitin-like proteins participate in diverse biological processes such as DNA repair, autophagy and signal transduction.

Animals↗

PubMed2003

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

Chromosomes, Bacterial↗

PubMed2003

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18.

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.

Adenosine Triphosphatases↗