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Genomics

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PubMed2001

Expressed sequence tags for the chicken genome from a normalized, ten-day-old white leghorn whole embryo cDNA library. 2. Comparative DNA sequence analysis of guinea fowl, quail, and turkey genomes.

Accelerated efforts to develop a high-utility chicken genome map have resulted in the development of resources that may be useful for genetic analysis in other economically important poultry species. Here we describe a total of 26 comparative genomic DNA sequences (CGS) for the guinea fowl, Japanese quail, and domestic turkey developed using 10 primer pairs specific for 10 previously reported, unique, chicken expressed sequence tags (EST). The total length of CGS developed for each of the three species was 4,193, 4,597, and 6,057 bp in quail, turkey, and guinea fowl, respectively. About 70% of the CGS showed significant sequence similarity to reference database sequences, including the reference chicken EST and other avian and nonavian genes. A majority of the between-species comparisons of the CGS from all but two primer pairs were significant and ranged from 81 to 99%. The percentage similarity of the CGS appears to be a function of phylogenetic relatedness and was generally higher for comparisons between the chicken, quail, and turkey and lower between the guinea fowl and chicken, quail, or turkey. Maximum likelihood estimation of the phylogenetic relationships using CGS from two primer pairs also showed a closer relationship, as expected, among chicken, quail, and turkey than between guinea fowl and either chicken, quail, or turkey. Within the guinea fowl, quail, and turkey CGS developed, the total number of single nucleotide polymorphisms detected was 28, 17, and 14, respectively. Together, these resources represent tools that will facilitate genetic analysis of species that have been studied very little and our understanding of their genomes and genome evolution.

Animals↗

PubMed1994

Cloning of the Nilaparvata lugens reovirus genome: conserved terminal nucleotide sequences and nucleotide sequence of genome segment S10.

The segmented double-stranded RNA genome of Nilaparvata lugens reovirus (NLRV) was cloned, and the nucleotide sequence of genome segment S10 and terminal nucleotide sequences of the rest of the segments were determined. Genome segment S10 of NLRV consisted of 1430 nucleotides with a single open reading frame extending for 1293 nucleotides from nucleotide 46. It encoded a polypeptide of 431 amino acids with an M(r) of 49.4K, which was a non-structural protein. The plus-strand RNA of all genome segments had the same conserved trinucleotide 5' AGU- and hexanucleotide-GUUGUC 3' in the 5'- and 3'-terminal regions, respectively. These conserved terminal sequences resembled those found in the segments of the members of the genus Fijivirus.

Amino Acid Sequence↗

PubMed2000

The Oenococcus oeni genome: physical and genetic mapping of strain GM and comparison with the genome of a 'divergent' strain, PSU-1.

The physical and genetic maps of the Oenococcus oeni strains GM and PSU-1, which represent two genomic divergent groups on the basis of macrorestriction and ribotyping analysis, were compared. To achieve this comparison, the GM maps were constructed and the PSU-1 maps, already established, were improved. All the recognition sites of the restriction enzymes ASC:I, I-CEU:I, FSE:I, NOT:I and SFI:I were located in both chromosomes and the position of 26 genetic markers, including two rrn operons and 14 new putative oenococcal genes, were allocated to the restriction fragments generated by the five enzymes. The comparative analysis of O. oeni GM and PSU-1 genomes revealed extensive conservation of loci order. As for the differences encountered in the locations of restriction sites, they seem to be a reflection of the differences in restriction fragment sizes, explainable by insertion/deletion events and point mutations. No evidence for major genomic rearrangements was found. The genomic conservation between the two strains is in agreement and suggests homogeneity within the species, which was not unexpected in view of the restricted ecological niche of O. oeni. Further comparisons of physical maps, both of O. oeni strains and related species, will certainly help to assess whether O. oeni is really an homogeneous species and physical mapping is suitable for taxonomic purposes, both at the supra- and intraspecific levels.

Chromosome Mapping↗

PubMed2002

Two-dimensional bacterial genome display: a method for the genomic analysis of mycobacteria.

Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.

Bacterial Proteins↗

PubMed2003

Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution.

Group Astreptococcus (GAS) is a gram-positive bacterial pathogen that causes various suppurative infections and nonsuppurative sequelae. Since the late 1980s, streptococcal toxic-shock like syndrome (STSS) and severe invasive GAS infections have been reported globally. Here we sequenced the genome of serotype M3 strain SSI-1, isolated from an STSS patient in Japan, and compared it with those of other GAS strains. The SSI-1 genome is composed of 1,884,275 bp, and 1.7 Mb of the sequence is highly conserved relative to strain SF370 (serotype M1) and MGAS8232 (serotype M18), and almost completely conserved relative to strain MGAS315 (serotype M3). However, a large genomic rearrangement has been shown to occur across the replication axis between the homologous rrn-comX1 regions and between two prophage-coding regions across the replication axis. Atotal of 1 Mb of chromosomal DNA is inverted across the replication axis. Interestingly, the recombinations between the prophage regions are within the phage genes, and the genes encoding superantigens and mitogenic factors are interchanged between two prophages. This genomic rearrangement occurs in 65% of clinical isolates (64/94) collected after 1990, whereas it is found in only 25% of clinical isolates (7/28) collected before 1985. These observations indicate that streptococcal phages represent important plasticity regions in the GAS chromosome where recombination between homologous phage genes can occur and result not only in new phage derivatives, but also in large chromosomal rearrangements.

Attachment Sites, Microbiological↗

PubMed2005

Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes.

The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.

Amino Acid Substitution↗

PubMed2002

Genomic signature tags (GSTs): a system for profiling genomic DNA.

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

Binding Sites↗

PubMed2006

Ubiquitous selective constraints in the Drosophila genome revealed by a genome-wide interspecies comparison.

Non-coding DNA comprises approximately 80% of the euchromatic portion of the Drosophila melanogaster genome. Non-coding sequences are known to contain functionally important elements controlling gene expression, but the proportion of sites that are selectively constrained is still largely unknown. We have compared the complete D. melanogaster and Drosophila simulans genome sequences to estimate mean selective constraint (the fraction of mutations that are eliminated by selection) in coding and non-coding DNA by standardizing to substitution rates in putatively unconstrained sequences. We show that constraint is positively correlated with intronic and intergenic sequence length and is generally remarkably strong in non-coding DNA, implying that more than half of all point mutations in the Drosophila genome are deleterious. This fraction is also likely to be an underestimate if many substitutions in non-coding DNA are adaptively driven to fixation. We also show that substitutions in long introns and intergenic sequences are clustered, such that there is an excess of substitutions <8 bp apart and a deficit farther apart. These results suggest that there are blocks of constrained nucleotides, presumably involved in gene expression control, that are concentrated in long non-coding sequences. Furthermore, we infer that there is more than three times as much functional non-coding DNA as protein-coding DNA in the Drosophila genome. Most deleterious mutations therefore occur in non-coding DNA, and these may make an important contribution to a wide variety of evolutionary processes.

Animals↗

PubMed2001

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea.

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Archaeal Proteins↗

PubMed2006

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region.

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

Cenchrus↗

PubMed2004

High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics.

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.

Animals↗

PubMed2004

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization.

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.

Base Sequence↗

PubMed1997

New insights into the genomic organization and origin of the major histocompatibility complex: role of chromosomal (genome) duplication in the emergence of the adaptive immune system.

Recently, it became clear that the human and mouse genomes contain at least three regions paralogous to the major histocompatibility complex (MHC) region. This observation led us to the proposal that the MHC region emerged as a result of chromosomal duplication that took place at an early stage of vertebrate evolution. Here I briefly review this proposal. Accumulating evidence indicates that (a) genome-wide duplication(s) took place close to the origins of vertebrates. Taking this and others into account, I suggest that the duplication(s) involving the MHC region probably took place as a part of the genome-wide duplication(s). The human T cell receptor (TCR) and immunoglobulin (Ig) genes also appear to be located on paralogous chromosomal segments. These findings raise the possibility that the genome-wide duplication provided a major impetus not only to the emergence of the full-fledged MHC system, but also to the appearance of other key molecules of the adaptive immune system such as TCR and Ig.

Animals↗

PubMed2006

Whole-genome sequence of Listeria welshimeri reveals common steps in genome reduction with Listeria innocua as compared to Listeria monocytogenes.

We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an F(o)F(1) ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of "fitness" genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.

Chromosomes, Bacterial↗

PubMed2003

The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.

The genome sequence of bacteriophage phiA1122 has been determined. phiA1122 grows on almost all isolates of Yersinia pestis and is used by the Centers for Disease Control and Prevention as a diagnostic agent for the causative agent of plague. phiA1122 is very closely related to coliphage T7; the two genomes are colinear, and the genome-wide level of nucleotide identity is about 89%. However, a quarter of the phiA1122 genome, one that includes about half of the morphogenetic and maturation functions, is significantly more closely related to coliphage T3 than to T7. It is proposed that the yersiniophage phiA1122 recombined with a close relative of the Y. enterocolitica phage phiYeO3-12 to yield progeny phages, one of which became the classic T3 coliphage of Demerec and Fano (M. Demerec and U. Fano, Genetics 30:119-136, 1945).

Amino Acid Sequence↗

PubMed2006

The genome of the novel phage Rtp, with a rosette-like tail tip, is homologous to the genome of phage T1.

A new Escherichia coli phage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity of rtp37, -38, -39, -41, -42, and -43 to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the lambda J protein, which determines lambda host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded by cor genes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much larger E. coli genome has only one such repeat sequence.

Amino Acid Sequence↗

PubMed1992

Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.

Sequencing of multiple recombinant clones generated from polymerase chain reaction-amplified products demonstrated that the degree of heterogeneity of two well-conserved regions of the hepatitis C virus (HCV) genome within individual plasma samples from a single patient was consistent with a quasispecies structure of HCV genomic RNA. About half of circulating RNA molecules were identical, while the remaining consisted of a spectrum of mutants differing from each other in one to four nucleotides. Mutant sequence diversity ranged from silent mutations to appearance of in-frame stop codons and included both conservative and nonconservative amino acid substitutions. From the relative proportion of essentially defective sequences, we estimated that most circulating particles should contain defective genomes. These observations might have important implications in the physiopathology of HCV infection and underline the need for a population-based approach when one is analyzing HCV genomes.

Amino Acid Sequence↗

PubMed1998

Deletion analysis of a defective interfering Semliki Forest virus RNA genome defines a region in the nsP2 sequence that is required for efficient packaging of the genome into virus particles.

The 1,244-nucleotide genome of Semliki Forest virus (SFV) defective interfering (DI) RNA 19 (DI-19) is coterminal with the infectious genome and contains two major deletions. One deletion removes the end of the nsP1 gene and the beginning of the nsP2 gene, and the other removes the end of the nsP2 gene, the nsP3 and nsP4 genes, and all of the structural protein genes (M. Thomson and N. J. Dimmock, Virology 199:354-365, 1994). Like all DI SFV RNAs, DI-19 contains three regions that are conserved. Region a comprises the 5' terminus continuous with part of the nsP1 gene, region b comprises a central part of the nsP2 gene, and region c comprises the 3' terminus and the associated untranslated region. A deletion analysis of the 265-nucleotide b region (nucleotides 679 to 943, inclusive) was undertaken to determine its role in genome replication and packaging into DI virus particles. Deleted plasmids were constructed and transcribed, and the resulting DI RNAs were transfected into SFV-infected BHK cells. Putative progeny DI virus particles that had been released into the tissue culture fluid were then serially passaged in new monolayers together with added high-multiplicity SFV, and cells and tissue culture fluids were tested for the presence of DI RNA by reverse transcription-PCR. DI RNA that had all of the b region deleted was replicated well in BHK-21 cells, as shown by the presence of large amounts of negative-sense DI RNA and an increase in the amount of positive-sense RNA in the cytoplasm, but was packaged very inefficiently, as indicated by very low amounts of DI RNA in the tissue culture fluid. The genome of a deletion mutant that retained the 3' 224 nucleotides of region b was packaged successfully, but one that retained only the 5' 41 nucleotides was not detected in the tissue culture fluid. These and other data suggest that nucleotides 720 to 777 of region b are of particular importance in the packaging process. This finding agrees with data obtained with Ross River virus and contrasts with the well-studied Sindbis alphavirus major packaging signal that is located within the nsP1 gene.

Animals↗