Biomedical subjects

T S Anantharaman

Publications and source records attributed to T S Anantharaman.

7 recordsLinked to original sources

PubMed2001

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Base Sequence↗

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

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

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

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

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↗

PubMed1997

Genomics via optical mapping. II: Ordered restriction maps.

In this paper, we describe our algorithmic approach to constructing ordered restriction maps based on the data created from the images of population of individual DNA molecules (clones) digested by restriction enzymes. The goal is to devise map-making algorithms capable of producing high-resolution, high-accuracy maps rapidly and in a scalable manner. The resulting software is a key component of our optical mapping automation tools and has been used routinely to map cosmid, lambda and BAC clones. The experimental results appear highly promising.

Algorithms↗