Showing papers by "J. Craig Venter Institute published in 2002"

Genome sequence of the human malaria parasite Plasmodium falciparum

Abstract: The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Abstract: Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences These are clustered into 33,409 'transcriptional units', contributing 901% of a newly established mouse transcriptome database Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome 41% of all transcriptional units showed evidence of alternative splicing In protein-coding transcripts, 79% of splice variations altered the protein product Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440

Abstract: Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Fast algorithms for large-scale genome alignment and comparison.

Abstract: We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory. The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system. It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes. A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences. We also describe a method to align more distantly related genomes by detecting protein sequence homology. This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches. This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another. The system code is being made freely available by the authors.

Whole-Genome Comparison of Mycobacterium tuberculosis Clinical and Laboratory Strains

Abstract: Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii

Abstract: Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.

The Genome of M. Acetivorans Reveals Extensive Metabolic and Physiological Diversity

Abstract: The Archaea remain the most poorly understood domain of life despite their importance to the biosphere. Methanogenesis, which plays a pivotal role in the global carbon cycle, is unique to the Archaea. Each year, an estimated 900 million metric tons of methane are biologically produced, representing the major global source for this greenhouse gas and contributing significantly to global warming (Schlesinger 1997). Methanogenesis is critical to the waste-treatment industry and biologically produced methane also represents an important alternative fuel source. At least two-thirds of the methane in nature is derived from acetate, although only two genera of methanogens are known to be capable of utilizing this substrate. We report here the first complete genome sequence of an acetate-utilizing (acetoclastic) methanogen, Methanosarcina acetivorans C2A. The Methanosarcineae are metabolically and physiologically the most versatile methanogens. Only Methanosarcina species possess all three known pathways for methanogenesis (Fig. ​(Fig.1)1) and are capable of utilizing no less than nine methanogenic substrates, including acetate. In contrast, all other orders of methanogens possess a single pathway for methanogenesis, and many utilize no more than two substrates. Among methanogens, the Methanosarcineae also display extensive environmental diversity. Individual species of Methanosarcina have been found in freshwater and marine sediments, decaying leaves and garden soils, oil wells, sewage and animal waste digesters and lagoons, thermophilic digesters, feces of herbivorous animals, and the rumens of ungulates (Zinder 1993). Figure 1 Three pathways for methanogenesis. Methanogenesis is a form of anaerobic respiration using a variety of one-carbon (C-1) compounds or acetic acid as a terminal electron acceptor. All three pathways converge on the reduction of methyl-CoM to methane (CH ... The Methanosarcineae are unique among the Archaea in forming complex multicellular structures during different phases of growth and in response to environmental change (Fig. ​(Fig.2).2). Within the Methanosarcineae, a number of distinct morphological forms have been characterized, including single cells with and without a cell envelope, as well as multicellular packets and lamina (Macario and Conway de Macario 2001). Packets and lamina display internal morphological heterogeneity, suggesting the possibility of cellular differentiation. Moreover, it has been suggested that cells within lamina may display differential production of extracellular material, a potential form of cellular specialization (Macario and Conway de Macario 2001). The formation of multicellular structures has been proposed to act as an adaptation to stress and likely plays a role in the ability of Methanosarcina species to colonize diverse environments. Figure 2 Different morphological forms of Methanosarcina acetivorans. Thin-section electron micrographs showing M. acetivorans growing as both single cells (center of micrograph) and within multicellular aggregates (top left, bottom right). Cells were harvested ... Significantly, powerful methods for genetic analysis exist for Methanosarcina species. These tools include plasmid shuttle vectors (Metcalf et al. 1997), very high efficiency transformation (Metcalf et al. 1997), random in vivo transposon mutagenesis (Zhang et al. 2000), directed mutagenesis of specific genes (Zhang et al. 2000), multiple selectable markers (Boccazzi et al. 2000), reporter gene fusions (M. Pritchett and W. Metcalf, unpubl.), integration vectors (Conway de Macario et al. 1996), and anaerobic incubators for large-scale growth of methanogens on solid media (Metcalf et al. 1998). Furthermore, and in contrast to other known methanogens, genetic analysis can be used to study the process of methanogenesis: Because Methanosarcina species are able to utilize each of the three known methanogenic pathways, mutants in a single pathway are viable (M. Pritchett and W. Metcalf, unpubl.). The availability of genetic methods allowing immediate exploitation of genomic sequence, coupled with the genetic, physiological, and environmental diversity of M. acetivorans make this species an outstanding model organism for the study of archaeal biology. For these reasons, we set out to study the genome of M. acetivorans.

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae

Abstract: The 2,160,267 bp genome sequence of Streptococcus agalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons among S. agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and to S. agalactiae. These in silico analyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V/R and 19 S. agalactiae strains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity among S. agalactiae strains, even of the same serotype, and provided insights into the evolution of virulence mechanisms.

The EcoCyc Database

Abstract: EcoCyc is an organism-specific pathway/genome database that describes the metabolic and signal-transduction pathways of Escherichia coli, its enzymes, its transport proteins and its mechanisms of transcriptional control of gene expression. EcoCyc is queried using the Pathway Tools graphical user interface, which provides a wide variety of query operations and visualization tools. EcoCyc is available at http://ecocyc.org/.

The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts

Abstract: The 3.31-Mb genome sequence of the intracellular pathogen and potential bioterrorism agent, Brucella suis, was determined. Comparison of B. suis with Brucella melitensis has defined a finite set of differences that could be responsible for the differences in virulence and host preference between these organisms, and indicates that phage have played a significant role in their divergence. Analysis of the B. suis genome reveals transport and metabolic capabilities akin to soil/plant-associated bacteria. Extensive gene synteny between B. suis chromosome 1 and the genome of the plant symbiont Mesorhizobium loti emphasizes the similarity between this animal pathogen and plant pathogens and symbionts. A limited repertoire of genes homologous to known bacterial virulence factors were identified.

Osteopontin identified as lead marker of colon cancer progression, using pooled Sample expression profiling

Abstract: Background: New tumor markers and markers of tumor progression are needed for improved staging and for better assessment of treatment of many cancers. Gene expression profiling techniques offer the opportunity to discover such markers. We investigated the feasibility of sample pooling strategy in combination with a novel analysis algorithm to identify markers. Methods: Total RNA from human colon tumors (n = 60) of multiple stages (adenomas; cancers with modified Astler Collier stages B, C, and D; and liver metastases) were pooled within stages and compared with pooled normal mucosal specimens (n = 10) by using oligonucleotide expression arrays. Genes that showed consistent increases or decreases in their expression through tumor progression were identified. Northern blot analysis was used to validate the findings. All statistical tests were two-sided. Results: More than 300 candidate tumor markers and more than 100 markers of tumor progression were identified. Northern analysis of 11 candidate tumor markers confirmed the gene expression changes. The gene for the secreted integrinbinding protein osteopontin was most consistently differentially expressed in conjunction with tumor progression. Its potential as a progression marker was validated (Spearman’s = 0.903; P<.001) with northern blot analysis using RNA from an independent set of 10 normal and 43 tumor samples representing all stages. Moreover, a statistically significant correlation between osteopontin protein expression and advancing tumor stage was identified with the use of 303 additional specimens (human cancer = 185, adenomas = 67, and normal mucosal specimens = 51) (Spearman’s = 0.667; P<.001). Conclusions: Sample pooling can be a powerful, cost-effective, and rapid means of identifying the most common changes in a gene expression profile. We identified osteopontin as a clinically useful marker of tumor progression by use of gene expression profiling on pooled samples. [J Natl Cancer Inst 2002;94:513–21]

Comparative Genome Sequencing for Discovery of Novel Polymorphisms in Bacillus anthracis

Abstract: Comparison of the whole-genome sequence of Bacillus anthracis isolated from a victim of a recent bioterrorist anthrax attack with a reference reveals 60 new markers that include single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats. Genome comparison detected four high-quality SNPs between the two sequenced B. anthracis chromosomes and seven differences among different preparations of the reference genome. These markers have been tested on a collection of anthrax isolates and were found to divide these samples into distinct families. These results demonstrate that genome-based analysis of microbial pathogens will provide a powerful new tool for investigation of infectious disease outbreaks.

The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium.

Abstract: The complete genome of the green-sulfur eubacterium Chlorobium tepidum TLS was determined to be a single circular chromosome of 2,154,946 bp. This represents the first genome sequence from the phylum Chlorobia, whose members perform anoxygenic photosynthesis by the reductive tricarboxylic acid cycle. Genome comparisons have identified genes in C. tepidum that are highly conserved among photosynthetic species. Many of these have no assigned function and may play novel roles in photosynthesis or photobiology. Phylogenomic analysis reveals likely duplications of genes involved in biosynthetic pathways for photosynthesis and the metabolism of sulfur and nitrogen as well as strong similarities between metabolic processes in C. tepidum and many Archaeal species.

Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000.

Abstract: The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.

Abstract: The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative σ factor, SigH

Abstract: The pathogenesis of tuberculosis involves multiple phases and is believed to involve both a carefully deployed series of adaptive bacterial virulence factors and inappropriate host immune responses that lead to tissue damage. A defined Mycobacterium tuberculosis mutant strain lacking the sigH-encoded transcription factor showed a distinctive infection phenotype. In resistant C57BL/6 mice, the mutant achieved high bacterial counts in lung and spleen that persisted in tissues in a pattern identical to those of wild-type bacteria. Despite a high bacterial burden, the mutant produced a blunted, delayed pulmonary inflammatory response, and recruited fewer CD4+ and CD8+ T cells to the lung in the early stages of infection. In susceptible C3H mice, the mutant again showed diminished immunopathology and was nonlethal at over 170 days after intravenous infection, in contrast to isogenic wild-type bacilli, which killed with a median time to death of 52 days. Complete genomic microarray analysis revealed that M. tuberculosis sigH may mediate the transcription of at least 31 genes directly and that it modulates the expression of about 150 others; the SigH regulon governs thioredoxin recycling and may be involved in the maintenance of intrabacterial reducing capacity. These data show that the M. tuberculosis sigH gene is dispensable for bacterial growth and survival within the host, but is required for the production of immunopathology and lethality. This phenotype demonstrates that beyond an ability to grow and persist within the host, M. tuberculosis has distinct virulence mechanisms that elicit deleterious host responses and progressive pulmonary disease.

Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14

Abstract: The mosquito-borne malaria parasite Plasmodium falciparum kills an estimated 0.7-2.7 million people every year, primarily children in sub-Saharan Africa. Without effective interventions, a variety of factors-including the spread of parasites resistant to antimalarial drugs and the increasing insecticide resistance of mosquitoes-may cause the number of malaria cases to double over the next two decades. To stimulate basic research and facilitate the development of new drugs and vaccines, the genome of Plasmodium falciparum clone 3D7 has been sequenced using a chromosome-by-chromosome shotgun strategy. We report here the nucleotide sequences of chromosomes 10, 11 and 14, and a re-analysis of the chromosome 2 sequence. These chromosomes represent about 35% of the 23-megabase P. falciparum genome.

Streptococcus pneumoniae proteins and nucleic acids

Abstract: The invention provides proteins and nucleic acid sequences from Streptocccus pneumoniae, together with a genome sequence. These are useful for the development of vaccines, diagnostics, and antibiotics.

A cut above: discovery of an alternative excision repair pathway in bacteria.

Abstract: Among the most highly conserved biochemical pathways in free living organisms are those involved in DNA repair (1). The ubiquitous pathway of nucleotide excision repair (NER) is responsible for the removal of environmentally induced DNA damage, such as the DNA lesions resulting from sunlight exposure or chemical carcinogens. Mutations or deficiencies in specific NER genes can lead to premature aging and cancer in humans (2, 3). The study of DNA repair in the bacterium Escherichia coli has helped us to understand the corresponding repair pathways in humans (4). NER can be viewed in four basic steps: (i) damage recognition and lesion verification; (ii) incision; (iii) excision; (iv) repair synthesis and ligation, as proposed nearly four decades ago. Damage recognition and verification are achieved by a protein machine that utilizes several components to sense a distortion in the double-helical duplex DNA. In E. coli the UvrA and UvrB proteins carry out these functions. If a putative lesion is identified by UvrA, the repair complex enlists the strand-opening activity of UvrB that helps to verify that the distortion is, in fact, due to a damaged nucleotide. It is believed that the beta-hairpin domain of UvrB is inserted into the DNA helix both to verify the damaged nucleotide and to establish which strand has been damaged (5–9). In both bacterial and eukaryotic species, strand opening and processing of the damage serves to further change the conformation of the DNA to help recruit nucleases to the lesion site to produce two endonucleolytic incisions in the phosphodiester backbone of the damaged strand, one on each side of the altered nucleotide(s). In E. coli, Bacillus caldotenax (9), and presumably in all other free living bacterial species, UvrB recruits the UvrC protein, which contains two functional endonuclease domains. The N-terminal part of this …

Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC.

Abstract: Previously, we described the generation and initial characterization of four Tn5 mutants of Pseudo- monas stutzeri strain KC with impaired ability to degrade carbon tetrachloride (Sepulveda-Torres et al., 1999). In this study, we show cloning and sequencing of an 8.3 kbp region in which all four transposons were located. This fragment encodes eight potential genes and is located in the central part of the 25 kbp fragment recently identified by Lewis et al .( 2000) and shown by them to be sufficient to confer carbon tetrachloride transfor- mation capability upon other pseudomonads. The four transposon insertion mutants mapped in ORF's F and I designated by Lewis et al .( 2000). This is consistent with the results by Lewis et al. (2000) that orfF is required for carbon tetrachloride degradation. We further established that orfI is required for CCl4 degradation since the three mutants in this ORF were unable to degrade carbon tetrachloride. We present our analysis of the gene and protein sequences from the 8.3 kbp region and propose a tentative model for the role of different genes in the synthesis and activity of pyridine-2,6-bis(thiocarboxylate) (PDTC), the secreted factor responsible for carbon tetrachlor- ide dechlorination. We also found a putative promoter that overlaps with a Fur-box-like sequence in the region upstream of mutated genes. To test this putative promoter region and Fur-box, we generated and ligated DNA fragments containing wild-type and mutant Fur-boxes to a lacZ reporter. The wild-type fragment showed promoter activity that is regulated by the concen- tration of iron in the medium. Finally, we screened a selection of Pseudomonas strains, including P. putida DSMZ 3601 - a strain known to produce PDTC - for the presence of the genes characterized in this study. None of the strains tested positive, suggesting that Pseudomonas stutzeri strain KC may possess a distinct biosynthetic pathway for PDTC production.

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis.

Abstract: Summary As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring colinearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

A preliminary comparison of the mouse and human genomes

Abstract: Accurate annotated assemblies of the mouse and human genomes enable a detailed comparison of the organization and evolution of the two genomes. We have completed several assemblies of both the mouse, with and without public data, and human genomes. Analysis of these assemblies suggests the mouse genome is about 10% smaller than the human genome primarily because of a difference in the content of repetitive DNA between the two genomes. More than 300,000 positions in these two genomes can be aligned with one another based on short segments of sequence similarity. These conserved segments significantly enhance the resolution of the resultant comparative maps and can be used to divide the genomes into regions of conserved-shared synteny. The genes found in such regions are highly conserved as is their relative order and orientation. Comparison of the human and mouse genome is expected to be key to deciphering the important biological information encoded in the mammalian genome. A prerequisite to comparing complex genomes such as those of mouse and human is the availability of annotated assemblies of both genomes that are comparable in quality and completeness. Since February 2001, we have assembled, annotated and delivered to our subscribers two versions of the human genome and two versions of the mouse genome. A third assembly of the human genome is being completed and will be delivered by fall of 2002. These annotated assemblies provide the starting materials for the genome-wide comparisons of the mouse and human reported here. We will begin with a description of the first Celera whole genome assembly of the mouse to provide a general basis of the quality and completeness of these data and then will report the results of a preliminary comparison between these two genomes.

Microarray analysis of the chondrocytic cell line T/C-28a2 under dynamic fluid shear

Abstract: The behavior of the chondrocytic cell line T/C-28a2 under shear flow was examined using a 32,448 element microarray. A parallel plate flow chamber was used to generate a shear stress level of 20 dyn/cm/sup 2/ for 1.5 or 24 hours (h), after which gene regulation was measured. Microarray analysis revealed differentially regulated genes affecting proliferation/differentiation, extracellular matrix/cytoskeleton, and inflammation at both time points. A ribonuclease protection assay was performed on a subset of genes to confirm the data obtained from the microarray. However, the cyclooxygenase-2 (COX-2) gene, which plays a role in the prostaglandin production in inflamed tissues and the synovium of rheumatoid arthritis (RA) patients, was studied further. Western hybridization revealed that COX-2 protein is present at 24 h, but not at 6 or 12 h. Also, immunofluorescence microscopy shows that COX-2 protein localizes in the cytosol after 24 h of shear, and is not present after 1.5 h. By examining the overall gene expression profiles of chondrocytes under different conditions of dynamic fluid shear, new insights on the pathogenesis of cartilage related diseases such as rheumatoid arthritis might be generated.