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SUMMARY: Recombination Detection Program (RDP) is a program that applies a pairwise scanning approach to the detection of recombination amongst a group of aligned DNA sequences. The software runs under Windows95 and combines highly automated screening of large numbers of sequences with a highly interactive interface for examining the results of the analyses.
Although most globins, including the N-terminal domains within chimeric proteins such as flavohemoglobins and globin-coupled sensors, exhibit a 3/3 helical sandwich structure, many bacterial, plant, and ciliate globins have a 2/2 helical sandwich structure. We carried out a comprehensive survey of globins in the genomes from the three kingdoms of life. Bayesian phylogenetic trees based on manually aligned sequences indicate the possibility of past horizontal globin gene transfers from bacteria to eukaryotes. blastp searches revealed the presence of 3/3 single-domain globins related to the globin domains of the bacterial and fungal flavohemoglobins in many bacteria, a red alga, and a diatom. Iterated psi-blast searches based on groups of globin sequences found that only the single-domain globins and flavohemoglobins recognize the eukaryote 3/3 globins, including vertebrate neuroglobins, alpha- and beta-globins, and cytoglobins. The 2/2 globins recognize the flavohemoglobins, as do the globin coupled sensors and the closely related single-domain protoglobins. However, the 2/2 globins and the globin-coupled sensors do not recognize each other. Thus, all globins appear to be distributed among three lineages: (i) the 3/3 plant and metazoan globins, single-domain globins, and flavohemoglobins; (ii) the bacterial 3/3 globin-coupled sensors and protoglobins; and (iii) the bacterial, plant, and ciliate 2/2 globins. The three lineages may have evolved from an ancestral 3/3 or 2/2 globin. Furthermore, it appears likely that the predominant functions of globins are enzymatic and that oxygen transport is a specialized development that accompanied the evolution of metazoans.
OBJECTIVE: To clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province. METHODS: Peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software. RESULTS: HIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42. CONCLUSION: The cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.
To determine if the emergence of hantavirus pulmonary syndrome in the United States was a consequence of recent amino-acid altering mutations and/or genetic reassortment with pathogenic hantaviruses, we examined lung and spleen tissues from seropositive deer mice trapped in August 1983 in Mono County, California, for hantaviral RNA by reverse transcriptase-directed polymerase chain reaction. Alignment and comparison of 1485 nucleotides of the S and M genomic segments enzymatically amplified from these tissues indicated that these deer mice harbored a hantavirus which was genetically similar, differing by less than 2% at the deduced amino-acid level, to the hantavirus implicated in cases of hantavirus pulmonary syndrome occurring in the Four-Corners region of New Mexico, Arizona, Utah, and Colorado in 1993. The peromyscine rodent-borne hantaviruses were, in turn, genetically distinct from other well-characterized hantaviruses, diverging by approximately 30% from Prospect Hill and Puumala viruses at the nucleotide and amino acid levels. Phylogenetic analysis using the maximum parsimony, neighbor-joining, and unweighted pair-group methods indicated that the Peromyscus-derived hantavirus shared a common ancestry with arvicolid rodent-borne hantaviruses. Overall congruency of the phylogenetic trees based on the S and M genomic sequences supported the evolutionary position of the peromyscine rodent-borne hantaviruses. Our data also establish the existence of a hantavirus enzootic in deer mice long before the recognition of hantavirus pulmonary syndrome in the United States.
Nucleotide sequence of the regulatory region R&R* and the partial 5'-end of the cnf1 gene (GenBank Accession No. AF023157) of Vibrio fischeri ATCC 7744 has been determined, and the cytotoxic necrotizing factor 1 (CNF1) encoded by the cnf1 gene is deduced. Alignment and comparison of the cytotoxic necrotizing factor 1s of V. fischeri and E. coli show that they are homologous. Nucleotide sequence reveals that the cnf1 gene is closely linked to the lux regulon in genome; the gene order of the cnf1 gene and the lux regulon is <--cnf1-R&R*<--rrn-<--luxR-R&R-luxI-luxC-luxD -luxA-luxB-luxE-luxG-omega-->, whereas R&R is the regulatory region of the lux regulon, and R&R* is the regulatory region of the cnf1 gene; the sequence approximately 2 kb lay between the luxR gene of the lux regulon and the cnf1 gene is an rrn-like operon. It is unexpected to find the cnf1 gene in V. fischeri, since the CNF1 protein enables necrosis; the marine luminous bacterium V. fischeri is never to be identified as a pathogenic microbe. The cnf1 gene might be concerned with symbosis of the luminous bacteria and host fishes.
The development of efficient DNA sequencing methods has led to the achievement of the DNA sequence of entire genomes from (to date) 55 prokaryotes, 5 eukaryotic organisms and 10 eukaryotic chromosomes. Thus, an enormous amount of DNA sequence data is available and even more will be forthcoming in the near future. Analysis of this overwhelming amount of data requires bioinformatic tools in order to identify genes that encode functional proteins or RNA. This is an important task, considering that even in the well-studied Escherichia coli more than 30% of the identified open reading frames are hypothetical genes. Future challenges of genome sequence analysis will include the understanding of gene regulation and metabolic pathway reconstruction including DNA chip technology, which holds tremendous potential for biomedicine and the biotechnological production of valuable compounds. The overwhelming volume of information often confuses scientists. This review intends to provide a guide to choosing the most efficient way to analyze a new sequence or to collect information on a gene or protein of interest by applying current publicly available databases and Web services. Recently developed tools that allow functional assignment of genes, mainly based on sequence similarity of the deduced amino acid sequence, using the currently available and increasing biological databases will be discussed.
The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.
Microtus is one of the most taxonomically diverse mammalian genera, including over 60 extant species. These rodents have evolved rapidly, as the genus originated less than 2 million years ago. If these numbers are taken at face value, then an average of 30 microtine speciation events have occurred every million years. One explanation for the rapid rate of cladogenesis in Microtus could be the karyotypic differentiation exhibited across the genus: diploid numbers range from 17 to 64. Despite the striking chromosomal variability within Microtus, phenotypic variation is unremarkable. To determine whether nucleotide substitution rates are also elevated in voles, we sequenced the entire mitochondrial DNA (mtDNA) genome of the Eurasian sibling vole (Microtus rossiaemeridionalis). We compared this genome to another previously sequenced vole mtDNA genome (Microtus kikuchii) and performed pairwise sequence comparisons with the mtDNA genomes of ten additional mammalian genera. We found that microtine mtDNA genomes are evolving more rapidly than any other mammalian lineage we sampled, as gauged by the rate of nucleotide substitution across the entire mtDNA genome as well as at each individual protein-coding gene. Additionally, we compared substitution rates within the cytochrome b gene to seven other rodent genera and found that Microtus mtDNA is evolving fastest. The root cause of accelerated evolution in Microtus remains uncertain, but merits further investigation.
Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.
The present work reported the cloning and characterization of two novel human genes--HN1 (hematopoietic- and neurologic-expressed sequence 1) and HN1L (HN1-like gene) which are proposed to be involved in embryo development. HN1 is mapped on chromosome 17q25.2, with two transcripts (1.0 and 1.6 kb in length, respectively) due to alternative splicing. HN1 is expressed abundantly in testis and skeletal muscle among 16 human tissues, and it is localized in the nucleus indicated by GFP fusion expression. Western blot confirmed that HN1 encodes a 16.5-kDa protein. HN1L is on chromosome 16p13.3, with three splicing in the length of 2.0, 4.0 and 4.2 kb, respectively. HN1L is expressed in a variety of tissues such as liver, kidney, prostate, testis and uterus at varying levels. HN1L gene encodes a 20-kDa protein, which is localized in both the nucleus and cytoplasm. Fourteen of HN1 and sixteen of HN1L homologous genes in different species were determined and analyzed by BLAST searches. Silicon analyses of the 14 orthologous proteins of HN1 and 16 orthologous proteins of HN1L revealed that they share great conservation in vertebrate. Additionally, we identified nine pseudogenes of HN1 (six) and HN1L (three) in the genomes of the human, mouse and rat. Based on sequence alignments and phylogenetic analysis, all these homologous genes and pseudogenes were defined as a HN1 gene family.
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A survey of gene sequences of species of Potyviridae, the most numerous family of plant viruses, has shown that the -GNNS- encoding region of the NIb gene is present in all c. 300 publicly available sequences of that region. We also report that relationships inferred from the sequence of the 1.6-2.1 kb portion of the genome between the -GNNS- encoding region and its 3' terminus reflect those of the remainder of the genome. Thus, the use for RT-PCR tests of the 'potyvirid primers' based on the -GNNS- encoding region and the 3' terminal poly-A region of the genome, will yield DNA fragments, the sequences of which are very likely to correctly identify viruses from which they were obtained.
The sequencing of the human genome has led to in silico predictions of far fewer genes than anticipated. Recent studies using serial analysis of gene expression have cast doubt on this finding. One study predicts the presence of many unidentified low abundance transcripts, whereas two others have mapped unique tags to previously unpredicted exons. Genome and transcriptome complexity is thus greater than predicted and many of the missing genes are probably expressed in low copy numbers or only in early embryonic tissues.
The human genome sequence provides a reference point from which we can compare ourselves with other organisms. Interspecies comparison is a powerful tool for inferring function from genomic sequence and could ultimately lead to the discovery of what makes humans unique. To date, most comparative sequencing has focused on pair-wise comparisons between human and a limited number of other vertebrates, such as mouse. Targeted approaches now exist for mapping and sequencing vertebrate bacterial artificial chromosomes (BACs) from numerous species, allowing rapid and detailed molecular and phylogenetic investigation of multi-megabase loci. Such targeted sequencing is complementary to current whole-genome sequencing projects, and would benefit greatly from the creation of BAC libraries from a diverse range of vertebrates.
Cadherins are components of adherens junctions and play critical roles during embryogenesis and organogenesis. They interact through the formation of anti-parallel dimers to mediate cell adhesion, migration and compaction. We recently showed that cadherins also play important roles in the inner ear; mutations in cadherin 23 (Cdh23) disrupt stereocilia organization on hair cells leading to deafness and vestibular dysfunction in waltzer mice. Here we extend our initial study on the structure and function of Cdh23. The mouse Cdh23 locus is comprised of two 5'-untranslated exons and 69 coding exons; together they cover a genomic distance of at least 350 kb. Amino acid sequence alignments and secondary structure prediction suggest that Cdh23 ectodomains adopt a conformation similar to the classic cadherins. Nucleotide sequence analysis of six alleles of waltzer reveals a strong correlation between loss of function mutations and the deafness/waltzing phenotype. A Cdh23 transcript with a spliced exon 68 is the predominantly expressed isoform in the organ of Corti. Age-related hearing loss (Ahl) is a non-syndromic trait in common inbred strains of mice associated with the Ahl locus on chromosome 10. Sequence comparison of Cdh23 between C57BL/6J and CAST/Ei identified ten amino acid polymorphisms. In the 5'- and 3'-untranslated regions we detected 11 single nucleotide polymorphisms. None of these sequence changes correlate with the Ahl phenotype. Our results provide the necessary framework for further characterization of Cdh23-related hearing loss in mice.
This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.
Denaturing HPLC was used to determine mutations occurring during the adaptive evolution of Escherichia coli K-12. The strains were evolved over 700 generations on glycerol as the sole carbon source from a sub-optimal to an optimal growth rate. The mutations detected by direct sequencing of amplicons of the glycerol-phosphate regulon repressor (glpR) gene were a synonymous substitution Val20Val in two separately evolved strains. Non-synonymous substitutions, Val119Gly and Gly179Trp, were also observed in each of the two strains. This procedure can be scaled to determine genome-scale sequence variations that have occurred during adaptive evolution.