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D C Schwartz

Publications and source records attributed to D C Schwartz.

At least 19 recordsLinked to original sources
PubMed1999

Whole-genome shotgun optical mapping of Deinococcus radiodurans.

A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.

Chromosomes, Bacterial↗
PubMed1999

Dynamics of DNA molecules in gel studied by fluorescence microscopy.

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis.

Chromosomes, Fungal↗
PubMed1999

Optical mapping of DNA polymerase I action and products.

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.

Bacteriophage lambda↗
PubMed1999

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

DNA Restriction Enzymes↗
PubMed1999

Optical mapping and its potential for large-scale sequencing projects.

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

Base Sequence↗
PubMed1999

A shotgun optical map of the entire Plasmodium falciparum genome.

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

Animals↗
PubMed1999

Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae.

The turnover of most mRNAs in Saccharomyces cerevisiae begins with deadenylation followed by decapping and 5'-->3' exonucleolytic digestion. An important question involves the mechanisms that allow particular mRNAs to exhibit different rates of both deadenylation and decapping. Since the cap structure plays a critical role in the assembly of translation initiation factors, we hypothesized that the status of the cytoplasmic cap binding complex would affect the rate of decapping. To test this hypothesis, we examined mRNA decay rates in yeast strains that were defective in several translation initiation factors that are part of the cap binding complex. These experiments yielded three significant observations. First, any mutation known to inhibit translation initiation also increased the rate of decapping. Second, decapping still occurred only after deadenylation, suggesting that the ability of the poly(A) tail to inhibit decapping does not require efficient translation of the transcript. Third, mutants with defects in translation initiation factors also showed an increase in the rate of deadenylation, suggesting that the rate of deadenylation may be controlled primarily by the translation status of the transcript. These results argue that the nature of the translation initiation complex is a critical factor in determining the mRNA half-life. This view also implies that some cis-acting sequences that modulate mRNA decay rate do so by affecting the translation status of the transcript.

Endoribonucleases↗
PubMed1999

Optical mapping of Plasmodium falciparum chromosome 2.

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.

Animals↗
PubMed1998

Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum.

Chromosome 2 of Plasmodium falciparum was sequenced; this sequence contains 947,103 base pairs and encodes 210 predicted genes. In comparison with the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, introns are more frequent, and proteins are markedly enriched in nonglobular domains. A family of surface proteins, rifins, that may play a role in antigenic variation was identified. The complete sequencing of chromosome 2 has shown that sequencing of the A+T-rich P. falciparum genome is technically feasible.

Amino Acid Sequence↗
PubMed1998

Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules.

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

Animals↗
PubMed1998

A quantitative study of optical mapping surfaces by atomic force microscopy and restriction endonuclease digestion assays.

Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.

DNA↗
PubMed1998

High-resolution restriction maps of bacterial artificial chromosomes constructed by optical mapping.

Large insert clone libraries have been the primary resource used for the physical mapping of the human genome. Research directions in the genome community now are shifting direction from purely mapping to large-scale sequencing, which in turn, require new standards to be met by physical maps and large insert libraries. Bacterial artificial chromosome libraries offer enormous potential as the chosen substrate for both mapping and sequencing studies. Physical mapping, however, has come under some scrutiny as being "redundant" in the age of large-scale automated sequencing. We report the development and applications of nonelectrophoretic, optical approaches for high-resolution mapping of bacterial artificial chromosome that offer the potential to complement and thereby advance large-scale sequencing projects.

Chromosome Mapping↗
PubMed1998

Mycobacterial genome structure.

Genome maps have been constructed for the mycobacterial pathogens Mycobacterium leprae and Mycobacterium tuberculosis, as well as for the attenuated vaccine strain Mycobacterium bovis BCG Pasteur. While the chromosomes of M. tuberculosis and M. bovis BCG Pasteur show extensive conservation at the gross level, comparison with M. leprae revealed a high degree of diversification, with a mosaic-like pattern apparent. The ordered libraries of M. tuberculosis and M. leprae produced during the course of these studies played a central role in the genome sequencing projects of these two bacilli, showing the utility of this approach for systematic sequencing of bacterial genomes.

Chromosome Mapping↗
PubMed1998

The large window ductus: a surgical trap.

A rare window type of patent ductus arteriosus is reported that was large (15 mm in maximal transverse dimension) but had virtually no length and hence was externally invisible. The smaller aortic isthmus (4 mm in diameter), which was intrapericardial, was mistaken for the ductus and was inadvertently clip-occluded, leading to death. After a specific diagnosis is made, the large window ductus should be patched on cardiopulmonary bypass with a transpulmonary approach.

Aorta, Thoracic↗
PubMed1998

Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet.

Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.

Bacteriophage lambda↗
PubMed1998

Effect of myocardial hypertrophy on systolic and diastolic function in children: insights from the force-frequency and relaxation-frequency relationships.

OBJECTIVE: The objective of this study was to evaluate the effect of myocardial hypertrophy on systolic and diastolic properties of the left ventricle in children. BACKGROUND: In children with myocardial hypertrophy, ejection phase indices are invariably increased. However, indices of force-generation, e.g., end-systolic elastance and invasive indices of diastolic properties, have been studied infrequently in children with myocardial hypertrophy. METHODS: We studied 10 children with congenital aortic stenosis or coarctation of aorta and nine control patients. Systolic properties were assessed from shortening fraction, end-systolic fiber elastance (Ef(es)) measured at resting heart rates, and force-frequency relationship measured at heart rates increasing from 110 to 160 beats per minute. Diastolic properties were assessed from time constant of relaxation (tau) at matched heart rates, chamber stiffness constant, myocardial stiffness constant, and relaxation-frequency relationship measured at gradually increasing heart rates. RESULTS: Ef(es) remained unchanged by myocardial hypertrophy, however, tau was prolonged (tauL: 27.3+/-2.3 vs. 21.8+/-2.2 ms, p < 0.001; and tauD: 43.2+/-3.1 vs. 34.3+/-3.3 ms, p < 0.001). Both chamber and myocardial stiffness constants remained unchanged. Incremental increases in heart rate produced incremental improvement in both contraction and relaxation. Slopes of force-frequency and relaxation-frequency relationships remained unchanged in the experimental group. However, the relaxation-frequency relationship manifested a parallel shift upward. CONCLUSIONS: In conscious, sedated children with myocardial hypertrophy, systolic function assessed by an index of force generation remains unchanged. However, relaxation is prolonged but passive diastolic properties remain unaffected. The combined effect of hypertrophy and heart rate does not alter the force-frequency and relaxation-frequency relationships.

Aortic Coarctation↗
PubMed1997

Is echocardiography or magnetic resonance imaging superior for precoarctation angioplasty evaluation?

We compared the dimensions of the aorta obtained by two-dimensional transthoracic echocardiography (echo) (median, 2.5 mo preangioplasty) and magnetic resonance imaging (MRI) (median, 4.2 mo preangioplasty) to those obtained by angiography (cath) in 13 patients (age, 7.7 +/- 1.6 yr; mean +/- SEM) who underwent evaluation for coarctation balloon angioplasty between April 1993-January 1996. Echo measurements were obtained from the suprasternal and subcostal sagittal planes, MRI measurements from axial and sagittal oblique views, and cath measurements from the straight lateral or oblique views. Measurements of the diameters of the aortic isthmus, coarctation, descending aorta at the diaphragm, and isthmus length were made by all three modalities. Presence of aorto-aortic collaterals was determined, and the coarctation length was delineated. Investigators were blinded to other measurement data prior to statistical analysis. Data analysis by repeated analysis of variance (ANOVA) and Student-Newman-Keuls testing revealed no statistically significant difference between systolic pressure gradient by clinical examination (32.2 +/- 5.9 mm Hg), peak instantaneous Doppler evaluation (37.5 +/- 2.9 mm Hg), or preangioplasty systolic pressure gradient (32.1 +/- 3.3 mm Hg). With the exception of measurements of the descending aorta (echo, 11.7 +/- 0.9 mm vs. MRI, 13.3 +/- 0.8 mm vs. cath, 14.0 +/- 1.3 mm; P = 0.04), there was no statistically significant difference in dimensions of the aortic isthmus (9.2 +/- 0.6 mm vs. 10.5 +/- 0.9 mm vs. 10.8 +/- 0.9 mm), coarctation site diameter (4.8 +/- 0.6 mm vs. 5.6 +/- 0.9 mm vs. 5.3 +/- 0.8 mm), or isthmus length (12.4 +/- 2.1 mm vs. 12.1 +/- 2.2 mm vs. 10.9 +/- 1.7 mm). The correlation coefficients derived from comparisons of MRI vs. cath to echo vs. cath were similar for all dimensions except for isthmus length (P < 0.01). MRI demonstrated aorto-aortic collaterals more frequently than echo, while echocardiography better demonstrated cardiac function and intracardiac anomalies. Both modalities detected the single patient with a long segment coarctation. We conclude that echocardiography and MRI provide similar definition of the coarcted thoracic aorta, and either test may be individually advantageous in specific clinical situations.

Adolescent↗