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PubMed2001

Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5'- and 3'-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5'-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/p210. Single amino acid changes within p195/p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.

Amino Acid Sequence↗

PubMed2005

Self-Organizing Map (SOM) unveils and visualizes hidden sequence characteristics of a wide range of eukaryote genomes.

Novel tools are needed for comprehensive comparisons of interspecies characteristics of massive amounts of genomic sequences currently available. An unsupervised neural network algorithm, Self-Organizing Map (SOM), is an effective tool for clustering and visualizing high-dimensional complex data on a single map. We modified the conventional SOM, on the basis of batch-learning SOM, for genome informatics making the learning process and resulting map independent of the order of data input. We generated the SOMs for tri- and tetranucleotide frequencies in 10- and 100-kb sequence fragments from 38 eukaryotes for which almost complete genome sequences are available. SOM recognized species-specific characteristics (key combinations of oligonucleotide frequencies) in the genomic sequences, permitting species-specific classification of the sequences without any information regarding the species. We also generated the SOM for tetranucleotide frequencies in 1-kb sequence fragments from the human genome and found sequences for four functional categories (5' and 3' UTRs, CDSs and introns) were classified primarily according to the categories. Because the classification and visualization power is very high, SOM is an efficient and powerful tool for extracting a wide range of genome information.

3' Untranslated Regions↗

PubMed2001

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Bacterial Proteins↗

PubMed2004

Little cherry virus-2: sequence and genomic organization of an unusual member of the Closteroviridae.

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

Amino Acid Sequence↗

PubMed1993

The rat adenine phosphoribosyltransferase sequence shows evolutionary rate variation among exons in rodents.

The complete genomic sequence of the rat APRT gene is described and compared with published mammalian sequences. The rat APRT gene organization is typical of other rodent APRTs with five exons, one large intron of 993 bp, and three smaller introns averaging 145 bp. Because complete sequences for mouse and Chinese hamster APRT are also known, it is possible to compare the evolutionary rates of change in the exons with those of the introns. The latter provide one possible estimate of underlying rates of change. It is shown that the APRT exons have differential rates of evolution in rodents and have had a recent and rapid burst of substitutions within the mouse lineage. Rates of change in the exons do not appear to be strongly correlated with the rates of change in the introns.

Adenine Phosphoribosyltransferase↗

PubMed1995

The tpa-1 gene of Caenorhabditis elegans encodes two proteins similar to Ca(2+)-independent protein kinase Cs: evidence by complete genomic and complementary DNA sequences of the tpa-1 gene.

The gene tpa-1 on chromosome IV of the nematode Caenorhabditis elegans plays a major and definitive role in the adversary action of tumour-promoting phorbol esters, which induce growth arrest and locomotory distress in the animal. The gene was deduced to code for a protein kinase C (PKC) homologue by molecular cloning. We have now sequenced the complete genomic and complementary DNAs for tpa-1 and have analysed their structural features in detail: (1) tpa-1 spans over 20 kb consisting of eleven exons and ten introns; (2) two different-sized mRNAs are generated from the tpa-1 locus; (3) both mRNAs are trans-spliced to the trans-spliced leader SL1; (4) both mRNAs encode PKC isoforms, which are most similar to Ca(2+)-independent novel PKC0; (5) the two PKC isoforms differ from each other in that the smaller lacks the amino-terminal region of the larger corresponding to the first four exons and a portion of the fifth exon; and (6) three introns are located at; identical positions in the polypeptide sequences aligned between the C. elegans tpa-1 product and a PKC of the fruit fly Drosophila melanogaster.

Amino Acid Sequence↗

PubMed2026

Whole-genome sequence of the type strain and two field strains of Arsenicicoccus dermatophilus causing pododermatitis in greater flamingos (Phoenicopterus roseus).

The complete genome sequence of the type strain KM894/11T and two field strains of Arsenicicoccus dermatophilus (KM18/12; KM9/12) isolated from foot skin lesions of captive greater flamingos was determined using Oxford Nanopore and Illumina sequencing technologies. The genomes differ structurally by a large ~650 kb inverted chromosomal fragment and plasmid content.

dermatitis↗

PubMed1999

Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803.

The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.

Amino Acid Sequence↗

PubMed2002

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain.

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Amino Acid Sequence↗

PubMed2006

inGeno--an integrated genome and ortholog viewer for improved genome to genome comparisons.

BACKGROUND: Systematic genome comparisons are an important tool to reveal gene functions, pathogenic features, metabolic pathways and genome evolution in the era of post-genomics. Furthermore, such comparisons provide important clues for vaccines and drug development. Existing genome comparison software often lacks accurate information on orthologs, the function of similar genes identified and genome-wide reports and lists on specific functions. All these features and further analyses are provided here in the context of a modular software tool "inGeno" written in Java with Biojava subroutines. RESULTS: InGeno provides a user-friendly interactive visualization platform for sequence comparisons (comprehensive reciprocal protein--protein comparisons) between complete genome sequences and all associated annotations and features. The comparison data can be acquired from several different sequence analysis programs in flexible formats. Automatic dot-plot analysis includes output reduction, filtering, ortholog testing and linear regression, followed by smart clustering (local collinear blocks; LCBs) to reveal similar genome regions. Further, the system provides genome alignment and visualization editor, collinear relationships and strain-specific islands. Specific annotations and functions are parsed, recognized, clustered, logically concatenated and visualized and summarized in reports. CONCLUSION: As shown in this study, inGeno can be applied to study and compare in particular prokaryotic genomes against each other (gram positive and negative as well as close and more distantly related species) and has been proven to be sensitive and accurate. This modular software is user-friendly and easily accommodates new routines to meet specific user-defined requirements.

Base Sequence↗

PubMed2003

GeneViTo: visualizing gene-product functional and structural features in genomic datasets.

BACKGROUND: The availability of increasing amounts of sequence data from completely sequenced genomes boosts the development of new computational methods for automated genome annotation and comparative genomics. Therefore, there is a need for tools that facilitate the visualization of raw data and results produced by bioinformatics analysis, providing new means for interactive genome exploration. Visual inspection can be used as a basis to assess the quality of various analysis algorithms and to aid in-depth genomic studies. RESULTS: GeneViTo is a JAVA-based computer application that serves as a workbench for genome-wide analysis through visual interaction. The application deals with various experimental information concerning both DNA and protein sequences (derived from public sequence databases or proprietary data sources) and meta-data obtained by various prediction algorithms, classification schemes or user-defined features. Interaction with a Graphical User Interface (GUI) allows easy extraction of genomic and proteomic data referring to the sequence itself, sequence features, or general structural and functional features. Emphasis is laid on the potential comparison between annotation and prediction data in order to offer a supplement to the provided information, especially in cases of "poor" annotation, or an evaluation of available predictions. Moreover, desired information can be output in high quality JPEG image files for further elaboration and scientific use. A compilation of properly formatted GeneViTo input data for demonstration is available to interested readers for two completely sequenced prokaryotes, Chlamydia trachomatis and Methanococcus jannaschii. CONCLUSIONS: GeneViTo offers an inspectional view of genomic functional elements, concerning data stemming both from database annotation and analysis tools for an overall analysis of existing genomes. The application is compatible with Linux or Windows ME-2000-XP operating systems, provided that the appropriate Java Runtime Environment is already installed in the system.

Bacterial Proton-Translocating ATPases↗

PubMed2006

Global epidemiology of HIV.

HIV is among the most generically variable of human pathogens. A comprehensive and detailed description of HIV strains in the pandemic is an important foundation for diagnosis, treatment, and prevention. The current sequence database for HIV includes almost 800 complete genome sequences, documenting HIV-1 groups M, O, and N, and HIV-2. Among HIV-1 group M strains, responsible for the vast majority of HIV infections worldwide, 743 sequences represent 9 genetic subtypes, 16 circulating recombinant forms (CRF) that are spreading in populations, and a variety of unique recombinant forms (URF), identified so far only from a single individual. The global distribution of HIV is complex and dynamic with regional epidemics harboring only a subset of the global diversity. HIV strains differ enormously in terms of global prevalence. Six strains account for the majority of HIV infections: HIV-1 subtypes A, B, C, D, and two of the CRF, CRF01-AE and CRF02_AG, respectively. Many of the known subtypes and recombinant forms are currently rare in the epidemic, but could spread more widely if favorable conditions arise. HIV-2 is largely restricted to West Africa at relatively low prevalence there. Groups O and N of HIV-1 are very rare in the pandemic. The goal of universal coverage of HIV-1 strains by diagnostic tests can be met by minimizing false negative test rates for the six globally prevalent HIV-1 group M strains and HIV-2, and by evaluating systematically coverage of rare subtypes and recombinant forms.

Genetic Variation↗

PubMed2004

Proteogenomic mapping as a complementary method to perform genome annotation.

The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence. Using recent advances in proteomics, we set out to predict the set of ORFs for an organism based principally on expressed protein-based evidence. Using a novel search strategy, we mapped peptides detected in a whole-cell lysate of Mycoplasma pneumoniae onto a genomic scaffold and extended these "hits" into ORFs bound by traditional genetic signals to generate a "proteogenomic map". We were able to generate an ORF model for M. pneumoniae strain FH using proteomic data with a high correlation to models based on sequence features. Ultimately, we detected over 81% of the genomically predicted ORFs in M. pneumoniae strain M129 (the originally sequenced strain). We were also able to detect several new ORFs not originally predicted by genomic methods, various N-terminal extensions, and some evidence that would suggest that certain predicted ORFs are bogus. Some of these differences may be a result of the strain analyzed but demonstrate the robustness of protein analysis across closely related genomes. This technique is a cost-effective means to add value to genome annotation, and a prerequisite for proteome quantitation and in vivo interaction measures.

Chromosome Mapping↗

PubMed2004

Serpins in unicellular Eukarya, Archaea, and Bacteria: sequence analysis and evolution.

Most serpins irreversibly inactivate specific serine proteinases of the chymotrypsin family. Inhibitory serpins are unusual proteins in that their native structure is metastable, and rapid conversion to a relaxed state is required to trap target enzymes in a covalent complex. The evolutionary origin of the serpin fold is unresolved, and while serpins in animals are known to be involved in the regulation of a remarkable diversity of metabolic processes, the physiological functions of homologues from other phyla are unknown. Addressing these questions, here we analyze serpin genes identified in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea, where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics of inhibitory serpins, and where multiple serpin genes are found in one genome, variability is displayed in the region of the reactive-center loop important for specificity. All the unicellular eukaryotic serpins have large hydrophobic or positively charged residues at the putative PI position. In contrast, none of the prokaryotic serpins has a residue of these types at the predicted P1 position, but many have smaller, neutral residues. Serpin evolution is discussed.

Amino Acid Sequence↗

PubMed2001

Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata.

Upon exposure to infection with digenetic trematodes such as Echinostoma paraensei, the freshwater snail Biomphalaria glabrata produces increased quantities of hemolymph lectins, some of which are unique polypeptides containing both immunoglobulin superfamily (IgSF) and fibrinogen domains. These unusual lectins have been termed fibrinogen-related proteins (FREPs), and recognize and precipitate digenean antigens. We here report 11 distinct FREP-encoding sequences from B. glabrata, and provide the complete genomic sequence for two of the most frequently recovered FREPs. The unique juxtaposition of IgSF and fibrinogen domains, previously known only from incomplete cDNAs, is confirmed. Sequences corresponding to known peptides derived from FREPs from hemolymph were found in one of these genes. Both genes contain four exons, the first encodes a putative signal peptide, the second and third a portion of an IgSF-type loop, and the fourth a fibrinogen domain. Cysteines, postulated to form an intrachain loop, are present in the IgSF domain and are separated from one another by 78 or 79 residues. The IgSF sequences most closely resemble V (variable)-type Ig domains, based on canonical and hydrophobic residues and predicted secondary structure. Some minor differences in genomic fragments isolated for each of the two sequences were noted and may represent allelic variants. The results may be of relevance in understanding the role of B. glabrata in transmission of Schistosoma mansoni, a digenean parasite that infects nearly 100 million people in the tropics.

Amino Acid Sequence↗

PubMed1999

A common mutation (epsilon1267delG) in congenital myasthenic patients of Gypsy ethnic origin.

OBJECTIVE: Mutation analysis of the acetylcholine receptor (AChR) epsilon subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). BACKGROUND: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR epsilon subunit (epsilon1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR epsilon subunit. METHODS: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for epsilon1267delG by PCR amplification and sequence analysis. RESULTS: The authors report the complete genomic sequence and organization of the gene coding for the epsilon subunit of the human AChR (accession number AF105999). Homozygous epsilon1267delG was identified in 13 CMS patients from 11 independent families. All epsilon1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. CONCLUSIONS: The mutation epsilon1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (epsilon1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease.

Adolescent↗

PubMed2004

Complete mitochondrial genome DNA sequence for two ophiuroids and a holothuroid: the utility of protein gene sequence and gene maps in the analyses of deep deuterostome phylogeny.

The complete mitochondrial genome sequences have been determined for the holothuroid Cucumaria miniata and two ophiuroid species Ophiopholis aculeata and Ophiura lütkeni. In addition, the nucleotide sequence of the mitochondrial protein-coding genes for the asteroid Pisaster ochraceus has been completed. Maximum-likelihood and LogDet distance analyses of concatenated protein-coding sequences produced a series of trees that did not conclusively support generally accepted models of echinoderm phylogeny. The ophiuroid data consistently demonstrated accelerated nucleotide divergence rates and lack of stationarity. This confounds the phylogenetic analyses. Molecular investigations using individual protein-coding gene alignments demonstrated that the cytochrome b gene exhibits the least deviation in rate and stationarity and generated some trees consistent with proposed echinoderm phylogenies. Phylogenies based on echinoderm mitochondrial gene rearrangements also proved problematic because of extensive variation in gene order between and within classes. A comparison of the two distinctive ophiuroid mitochondrial gene orders supports the hypothesis that O. lütkeni has a more derived mitochondrial gene order versus O. aculeata. The variation in the echinoderm mitochondrial gene maps reinforces the limitations of the application of mitochondrial gene rearrangements as a global phylogenetic tool.

Animals↗

PubMed2003

A genome sequence of novel SARS-CoV isolates: the genotype, GD-Ins29, leads to a hypothesis of viral transmission in South China.

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.

Base Sequence↗