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PubMed2004

Complete genomic nucleotide sequence of the temperate bacteriophage Aa Phi 23 of Actinobacillus actinomycetemcomitans.

The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced. Linear DNA contained in the phage particles is circularly permuted and terminally redundant. Therefore, the physical map of the phage genome is circular. Its size is 43,033 bp with an overall molar G+C content of 42.5 mol%. Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift. A putative function could be assigned to 23 of them. Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23. The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages. However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system. The attachment sites of Aa Phi 23 (attP) and several A. actinomycetemcomitans hosts (attB) are 49 bp long.

Aggregatibacter actinomycetemcomitans↗
PubMed2006

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades.

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

Base Sequence↗
PubMed1997

Complete sequence and genomic analysis of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.

Amino Acid Sequence↗
PubMed2006

Complete plastid genome sequences of Drimys, Liriodendron, and Piper: implications for the phylogenetic relationships of magnoliids.

BACKGROUND: The magnoliids with four orders, 19 families, and 8,500 species represent one of the largest clades of early diverging angiosperms. Although several recent angiosperm phylogenetic analyses supported the monophyly of magnoliids and suggested relationships among the orders, the limited number of genes examined resulted in only weak support, and these issues remain controversial. Furthermore, considerable incongruence resulted in phylogenetic reconstructions supporting three different sets of relationships among magnoliids and the two large angiosperm clades, monocots and eudicots. We sequenced the plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales), and used these data in combination with 32 other angiosperm plastid genomes to assess phylogenetic relationships among magnoliids and to examine patterns of variation of GC content. RESULTS: The Drimys, Liriodendron, and Piper plastid genomes are very similar in size at 160,604, 159,886 bp, and 160,624 bp, respectively. Gene content and order are nearly identical to many other unrearranged angiosperm plastid genomes, including Calycanthus, the other published magnoliid genome. Overall GC content ranges from 34-39%, and coding regions have a substantially higher GC content than non-coding regions. Among protein-coding genes, GC content varies by codon position with 1st codon > 2nd codon > 3rd codon, and it varies by functional group with photosynthetic genes having the highest percentage and NADH genes the lowest. Phylogenetic analyses using parsimony and likelihood methods and sequences of 61 protein-coding genes provided strong support for the monophyly of magnoliids and two strongly supported groups were identified, the Canellales/Piperales and the Laurales/Magnoliales. Strong support is reported for monocots and eudicots as sister clades with magnoliids diverging before the monocot-eudicot split. The trees also provided moderate or strong support for the position of Amborella as sister to a clade including all other angiosperms. CONCLUSION: Evolutionary comparisons of three new magnoliid plastid genome sequences, combined with other published angiosperm genomes, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots.

Base Composition↗
PubMed2003

In silico proteome analysis to facilitate proteomics experiments using mass spectrometry.

Proteomics experiments typically involve protein or peptide separation steps coupled to the identification of many hundreds to thousands of peptides by mass spectrometry. Development of methodology and instrumentation in this field is proceeding rapidly, and effective software is needed to link the different stages of proteomic analysis. We have developed an application, proteogest, written in Perl that generates descriptive and statistical analyses of the biophysical properties of multiple (e.g. thousands) protein sequences submitted by the user, for instance protein sequences inferred from the complete genome sequence of a model organism. The application also carries out in silico proteolytic digestion of the submitted proteomes, or subsets thereof, and the distribution of biophysical properties of the resulting peptides is presented. proteogest is customizable, the user being able to select many options, for instance the cleavage pattern of the digestion treatment or the presence of modifications to specific amino acid residues. We show how proteogest can be used to compare the proteomes and digested proteome products of model organisms, to examine the added complexity generated by modification of residues, and to facilitate the design of proteomics experiments for optimal representation of component proteins.

Journal Article↗
PubMed2003

Molecular resolution of a complex of potyviruses infecting solanaceous crops at the centre of origin in Peru.

Peru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20-30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.

Capsicum↗
PubMed2004

Genome sequence and attenuating mutations in West Nile virus isolate from Mexico.

The complete genome sequence of a Mexican West Nile virus isolate, TM171-03, included 46 nucleotide (0.42%) and 4 amino acid (0.11%) differences from the NY99 prototype. Mouse virulence differences between plaque-purified variants of TM171-03 with mutations at the E protein glycosylation motif suggest the emergence of an attenuating mutation.

Animals↗
PubMed2000

Genomic organization and gene function in Leishmania.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Animals↗
PubMed2001

Deciphering Campylobacter jejuni cell surface interactions from the genome sequence.

The completion of the Campylobacter jejuni genome sequence is a landmark in Campylobacter research. Discoveries directly arising from these data include the identification of a capsular polysaccharide, extensive capacity for phase variable gene expression and lipo-oligosaccharide structural phase variation. The recent identification of a unique system of general protein glycosylation in C. jejuni, a C. jejuni protein that is translocated into eukaryotic cells, and plasmid-encoded components of a putative type IV secretion system are likely to be significant in terms of the host-pathogen interaction.

Antigenic Variation↗
PubMed1997

Codon-anticodon assignment and detection of codon usage trends in seven microbial genomes.

We have assigned codon-anticodon recognition patterns for the whole set of transfer RNAs of Haemophilus influenzae Rd, Methanococcus jannaschii, and Synechocystis sp. PCC6803 using sequence information derived from the complete genome sequence of these organisms and have tabulated them along with those previously reported for Escherichia coli, Mycoplasma genitalium, Mycoplasma pneumoniae, and Saccharomyces cerevisiae. Using the resulting codon-anticodon tables, the bias in codon usage of genes encoding the entire protein and ribosomal protein complement of each of the seven microbial genomes was analyzed. Then, the codon adaptation index (CAIrp) for each protein gene was calculated using the codon usage preference of the ribosomal protein genes of the corresponding organism. Of the seven genomes examined, six showed CAIrp scores that roughly coincided with the expected level of gene expression. The result demonstrates that CAIrp analysis may be useful for prediction of the expression level of unknown genes when all or at least considerable portions of the genome sequence are available.

Codon↗
PubMed2026

Detection of feline kobuvirus in filtered fecal inocula and molecular epidemiological characterization of feline kobuvirus in Japan.

Feline kobuvirus (FKoV) is an enteric virus detected in cats in several countries, but its prevalence and genomic characteristics in Japan remain unclear. Here, we report the detection of FKoV in specific-pathogen-free (SPF) cats inoculated with filtered fecal material that had tested negative for FKoV by an initial RT-PCR assay. The inoculum was derived from a shelter-housed cat positive for a feline norovirus (FNoV) strain (KU22; GVI.1; GenBank accession no. LC935948), which shared 99% VP1 nucleotide identity with the Japanese strain M49-1. FKoV RNA was undetectable in all four inoculated cats at day 0 using a degenerate universal kobuvirus primer set, but was detected in all four cats at days 5, 20, and 30 post inoculation. FKoV RNA in the inoculum was negative by the initial RT-PCR assay but positive by the degenerate universal kobuvirus primer set, indicating that FKoV was present in the inoculum but was missed by the initial assay; FKoV RNA was subsequently detected in the inoculated cats, consistent with inoculum-associated introduction of FKoV. Near-complete genome sequencing confirmed that the Japanese strain belonged to the feline kobuvirus lineage, representing the first near-complete FKoV genome sequence from Japan. In a field survey of 83 domestic cats, FKoV RNA was detected in 10 samples (12.0%), with higher prevalence in cats with gastroenteritis than in apparently healthy cats (40.0% vs. 8.2%; Bonferroni-adjusted p = 0.049), and exclusively in cats younger than 1 year. These findings provide genomic and epidemiological data on FKoV in Japan and highlight the need for broad-spectrum screening of biological inocula used in experimental infection studies.

Animals↗
PubMed2003

Expressed sequence tags: alternative or complement to whole genome sequences?

Over three million sequences from approximately 200 plant species have been deposited in the publicly available plant expressed sequence tag (EST) sequence databases. Many of the ESTs have been sequenced as an alternative to complete genome sequencing or as a substrate for cDNA array-based expression analyses. This creates a formidable resource from both biodiversity and gene-discovery standpoints. Bioinformatics-based sequence analysis tools have extended the scope of EST analysis into the fields of proteomics, marker development and genome annotation. Although EST collections are certainly no substitute for a whole genome scaffold, this "poor man's genome" resource forms the core foundations for various genome-scale experiments within the as yet unsequenceable plant genomes.

Computational Biology↗
PubMed2005

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis.

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.

Bacterial Proteins↗
PubMed2003

Sequence mapping of the Californian MSW strain of Myxoma virus.

Partial sequence mapping of the MSW Californian strain of Myxoma virus was performed by cloning EcoRI and SalI restriction fragments of viral DNA and sequencing the ends of these. In this way, regions of 74 MSW open reading frames were sequenced and mapped onto the complete genome sequences of the related leporipoxviruses South American Myxoma virus and Rabbit fibroma virus to form a partial map of the MSW strain. In general, gene locations and sequences were conserved between the three viruses. However the Californian Myxoma virus was more closely related to South American myxoma virus than to Rabbit fibroma virus based on sequence comparisons and the presence of three genes that have been lost from the Rabbit fibroma virus genome. Compared to the other two viruses, the main difference found in the MSW genome was that the terminal inverted repeats were extended with the duplication of 5 complete open reading frames (M151R, M152R, M153R, M154L, M156R) and partial duplication of one open reading frame (M150R). This rearrangement was associated with the loss of the majority of the M009L open reading frame. Three known virulence genes, including the serine proteinase inhibitor (SERPIN) genes M151R and M152R and leukemia associated protein (LAP) gene M153R, and the potential virulence gene M156R are now present in two copies.

Animals↗