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At least 919 records · Page 51Linked to original sources
PubMed2003

Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling.

Despite extensive primary sequence diversity, crystal structures of several bacterial cytochrome P450 monooxygenases (P450s) and a single eukaryotic P450 indicate that these enzymes share a structural core of alpha-helices and beta-sheets and vary in the loop regions contacting individual substrates. To determine the extent to which individual structural features are conserved among divergent P450s existing in a single biosynthetic pathway, we have modeled the structures of four highly divergent P450s (CYP73A5, CYP84A1, CYP75B1, CYP98A3) in the Arabidopsis phenylpropanoid pathway synthesizing lignins, flavonoids and anthocyanins. Analysis of these models has indicated that, despite primary sequence identities as low as 13%, the structural cores and several loop regions of these P450s are highly conserved. Substrate docking indicated that all four enzymes employ a common strategy to identify their substrates in that their cinnamate-derived substrates align along helix I with their aromatic ring positioned towards the C-terminus of this helix and their aliphatic tails positioned towards the N-terminus. Further similarity was observed in the way the substrates contact the consensus P450 substrate recognition sites (SRS). Residues predicted to contact the aromatic ring region exist in SRS5, SRS6 and the C-terminal portion of SRS4 and residues contacting the distal end of each substrate exist in SRS1, SRS2 and the N-terminal portion of SRS4. Alignments of the regions contacting the aromatic ring region indicate that SRS4, SRS5 and SRS6 share higher degrees of sequence conservation than found in SRS1, SRS2 or the full-length protein.

Amino Acid Sequence↗
PubMed1993

Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis.

The capacities to induce the synthesis of hepatitis B virus (HBV) unit-length DNA were compared for two HBV DNAs with an overall sequence diversity of about 10%. They had been cloned from serum (DNA2) and from a hepatocellular carcinoma (DNA4), respectively. As a major difference, DNA4 carries a translational stop signal preventing the synthesis of precore protein. Progeny DNA yields obtained after transfection with respective pregenome transcription units allocated DNA2 to a low-replicator and DNA4 to a high-replicator phenotype. Cotransfection of DNA2 interfered with progeny DNA synthesis induced by DNA4. By mutual exchange of restriction fragments, the region on the viral genome responsible for the differing replicator phenotypes was confined to a sequence comprising the 3'-terminal part of the X gene, core promoter, encapsidation signal epsilon, precore/core gene, and 5'-terminal part of the pol gene. Point mutations in DNA2 abolishing proper expression of the precore gene strongly enhanced the yield of progeny DNA, whereas cotransfection of a precore expression plasmid with DNA4 or with the mutated DNA2 substantially lowered the amount of progeny DNA. Hence, precore expression acts as an inhibitory principle for HBV replication. The same stop mutation as in DNA4 has been found to arise frequently in virus carriers. Loss of precore expression and concomitant conversion to a more severe hepatitis, as observed in the course of a chronic infection, thus can be explained by a relaxation of replication-level control.

DNA, Viral↗
PubMed1990

Segregation of mitochondrial genomes in a heteroplasmic lineage with Leber hereditary optic neuroretinopathy.

Relatively little is known about the factors maintaining mitochondrial DNA (mtDNA) sequence diversity in humans. A detailed understanding of the transmission genetics of mtDNA has been partly hampered by the lack of evidence for heteroplasmic individuals. Among families with Leber hereditary optic neuroretinopathy, we found a maternal lineage with individuals heteroplasmic for a single nucleotide change, and we were able to follow the segregation of polymorphic mitochondrial genomes over 3 generations. The results show that rapid segregation can occur but also that the level of heteroplasmy can be maintained from one generation to another. In this family the disease phenotype is associated with the mtDNA sequence change, confirming the involvement of the mutation in the disease.

Base Sequence↗
PubMed2004

Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae.

Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection.

Base Composition↗
PubMed2006

Geographic ranges, population structure, and ages of sexual and parthenogenetic snail lineages.

Asexual reproduction is thought to doom organisms to extinction due to mutation accumulation and parasite exploitation. Theoretical models suggest that parthenogens may escape the negative effects of conspecifics and biological enemies through escape in space. Through intensive sequencing of a mitochondrial DNA (mtDNA) and a nuclear intron locus in sexual and parthenogenetic freshwater snails (Campeloma), I examine three questions: (1) Are sexual mtDNA lineages more restricted geographically than parthenogenetic mtDNA lineages? (2) Are independent parthenogenetic lineages shorter lived than sexual lineages? and (3) Do parthenogens have higher intraindividual nuclear sequence diversity and form well-differentiated monophyletic groups as expected under the Meselson effect? Geographic ranges of parthenogenetic lineages are significantly larger than geographic ranges of sexual lineages. Based on coalescence times under different demographic assumptions, asexual lineages are short lived, but there is variation in clonal ages. Although alternative explanations exist, these results suggest that asexual lineages may persist in the short term through dispersal, and that various constraints may cause geographic restriction of sexual lineages. Both allotriploid and diploid Campeloma parthenogens have significantly higher allelic divergence within individuals, but show limited nuclear sequence divergence from sexual ancestors. In contrast to previous allozyme evidence for nonhybrid origins of diploid Campeloma parthenogens, cryptic hybridization may account for elevated heterozygosity.

Aging↗
PubMed2003

Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences.

The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.

Aeromonas↗
PubMed2005

A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame.

BACKGROUND: Hepatitis C virus (HCV) is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR) of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile. RESULTS: RT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF) from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon) for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments. CONCLUSION: Optimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

DNA, Complementary↗
PubMed2005

Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form.

Animals↗
PubMed2001

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Amino Acid Sequence↗
PubMed2001

A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Animals↗
PubMed1995

Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization.

Application of reverse transcription (RT)-PCR to detect small round-structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polymerase gene of SRSVs belonging to two distinct genogroups represented by Norwalk virus (primers G-1) and Snow Mountain agent (primers G-2). All 22 SRSV strains examined that had been classified previously by solid-phase immune electron microscopy into four antigenic types (UK1, UK2, UK3, and UK4) could be detected by RT-PCR with these two primer sets. The G-1 primer set detected 6 UK2 strains, and the G-2 primers detected 16 strains, including 7 UK1, 5 UK3, and 4 UK4 strains. On the basis of nucleotide sequences of 81-bp fragments of the RT-PCR products from 13 strains determined in this study, together with those previously reported for 17 SRSV strains, we designed four sets of internal oligonucleotide probes (P1-A, P1-B, P2-A, and P2-B) for Southern hybridization, using chemiluminescent detection. The P1-A probe hybridized with PCR products from the UK2 strains; the P1-B probe, with products from two of the seven UK1 strains; the P2-A probe, with four of the remaining five UK1 strains; and the P2-B probe, with products from both UK3 and UK4 strains, as well as with one strain originally typed as UK1 which showed cross-reactivity with UK4 upon retesting by solid-phase immune electron microscopy. RT-PCR with both the G-1 and the G-2 primer sets can increase the detection rate of the many antigenically distinct SRSVs and, when combined with Southern hybridization, may predict the antigenic type of the SRSV associated with infection.

Adult↗
PubMed1996

Longitudinal studies of viral sequence, viral phenotype, and immunologic parameters of human immunodeficiency virus type 1 infection in perinatally infected twins with discordant disease courses.

Perinatal human immunodeficiency virus type 1 (HIV-1) infections cause a broad spectrum of clinical disease and are variable in both the age of the patient at onset of serious disease and the progression of the clinical course. Heterozygotic perinatally infected twins with a marked difference in their clinical courses were monitored during the first 2 years of life. Twin B, the second-born twin, developed AIDS by 6 months of age and died at 22 months of age, while twin A remained minimally symptomatic through the first 2 years. Sequential blood specimens were obtained from the twins in order to characterize the immunologic properties of the children and the phenotypes and genotypes of the HIV-1 isolates at various times. Twin A developed neutralizing antibodies and a high-level antibody-mediated cellular cytotoxicity (ADCC) response, while twin B had no neutralizing antibody and a much lower ADCC response. The virus isolates obtained from the two children at various time points proliferated equally well in peripheral blood mononuclear cells, were nonsyncytium inducing, and could not infect established T-cell lines. They differed in their ability to infect primary macrophages. In parallel to the biological studies, the HIV-1 tat and part of the env gene sequences of the longitudinal isolates at four time points were determined. Sequences of virus from both twins at different time points were highly conserved; the viruses evolved at a similar rate until the last analyzed time point, at which there was a dramatic increase in sequence diversity for the sicker child, especially in the tat gene. Our results show that the viruses isolated at different times do not have significant changes in growth properties. The absence or low levels of neutralizing antibodies may correlate with disease progression in the twins.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Clinical role for a superantigen in Yersinia pseudotuberculosis infection.

Yersinia pseudotuberculosis is an enteric pathogen that causes a variety of clinical symptoms in the human. Recently, we reported the production of a superantigen (Y. pseudotuberculosis-derived mitogen, YPM) by this organism and characterized the gene structure of ypm. To further study the potential pathogenic role of YPM in Y. pseudotuberculosis infection, we assayed IgG anti-YPM antibodies and T cell antigen receptor-Vbeta expression of the T cells in peripheral blood and in mesenteric lymph node in patients acutely infected with Y. pseudotuberculosis. 20 out of 33 patients (61%) had an elevated antibody titer compared with healthy controls (P = 0.0001). Patients with systemic symptoms such as lymphadenopathy, transient renal dysfunction, and arthritis had significantly higher titers of anti-YPM than patients with gastrointestinal tract symptoms alone. T cells bearing the Vbeta3 gene segment were significantly increased (P = 0.009) among acute phase patients compared with healthy children. During the convalescence phase of the illness, there was a reduction in the abnormal level of Vbeta3 T cells. Moreover, in the mesenteric lymph node, an elevated level of Vbeta3 T cells compared with peripheral blood and a sequence diversity in the junctional region of the T cell antigen receptor beta-chain containing Vbeta3 element was observed in one patient. Together, these findings suggest that YPM was produced in vivo and played an important role in the pathogenesis of Y. pseudotuberculosis infection.

Adolescent↗
PubMed1998

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis.

DNA of thirteen haemolytic Proteus penneri strains of clinical origin, all producing calcium dependent haemolysin and having been derived from four European countries was examined for plasmid profile, and outer membrane protein profile, by random amplified polymorphic DNA-PCR (RAPD-PCR) method, and digestions with restriction endonucleases were performed. All strains contained two large plasmids of approximately 60 and 70 kilobase pairs (kb). In addition, four strains contained a small plasmid of about 6 kb. These four strains produced cell-bound haemolysin only. Outer membrane protein analysis revealed subtle differences between strains. RAPD-PCR with primer I (CCGCAGCCAA) revealed 13 types, whereas primer II (AACGCGCAAC) yielded only two main types of different patterns. Results with primer I suggests a DNA sequence diversity within this species. The RAPD-PCR method provides a fast, economical and reproducible means for the typing of P. penneri. Digestion with restriction endonucleases indicated a high level of DNA methylation in this species.

Bacterial Outer Membrane Proteins↗
PubMed2006

Domestication history in the Medicago sativa species complex: inferences from nuclear sequence polymorphism.

DNA sequence polymorphism carries genealogical information and allows for testing hypotheses on selection and population history, especially through coalescent-based analysis. Understanding the evolutionary forces at work in plant domestication and subsequent selection is of critical importance for the management of genetic resources. In this study, we surveyed DNA sequence diversity at two assumed neutral nuclear loci in the wild-domesticated species complex of alfalfa (Medicago sativa L.). A high level of polymorphism was detected. The domesticated pool contains on average 31% less diversity than the wild pool, but with a high heterogeneity among loci. Coalescent simulations of the domestication process showed that this result cannot be explained by assuming a constant population size but is rather consistent with a demographic bottleneck during domestication. A very low level of divergence was detected between the wild and the domesticated forms as well as between the related subspecies of the M. sativa species complex. However, the originality of the Spanish wild populations, already observed based on mitochondrial DNA polymorphism, was confirmed. These results, together with patterns of intrapopulation polymorphism, suggest that nuclear sequence polymorphism could be a promising tool, complementary to mitochondrial DNA and phenotypic evaluations, to investigate historical demographic and evolutionary processes.

Biological Evolution↗
PubMed2003

What is Babesia microti?

Babesia microti (Apicomplexa: Piroplasmida) has historically been considered a common parasite of Holarctic rodents. However, human babesiosis due to this species has generally been limited to the northeastern seaboard of the United States and Minnesota and Wisconsin. The absence of reports of B. microti babesiosis from sites where the agent is enzootic, such as in western Europe, remains unexplained. Previous work focusing on the 18S rDNA demonstrates little sequence diversity among samples from allopatric host populations across a wide geographical area. It may be that genetic diversity is underestimated due to sample size or the gene analysed. Accordingly, we collected blood or spleen samples from American or Eurasian animals with parasites that were morphologically consistent with B. microti, amplified the 18S rDNA and beta-tubulin gene, and conducted phylogenetic analysis. Surprisingly, what was considered to be 'B. microti' by microscopy appears to be a diverse species complex. We identify 3 distinct clades within this complex, including parasites from non-rodent hosts. Rodent parasites comprise 2 clades, one representing zoonotic isolates, and the other apparently maintained in microtine rodents, and therefore their morphological detection within animals from a site does not necessarily imply a risk to public health.

Animals↗
PubMed2002

Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors.

Seven-transmembrane-domain proteins encoded by the vomeronasal receptor V1r and V2r gene superfamilies, and expressed by vomeronasal sensory neurons, are believed to be pheromone receptors in rodents. Four V1r gene families have been described in the mouse (V1ra, V1rb, V1rc and V3r). Here we have screened near-complete mouse genomic databases to obtain a first global draft of the mouse V1r repertoire, including 104 new V1r genes. It comprises eight new and extremely isolated families in addition to the four families previously identified. Members of these new families were expressed in vomeronasal sensory neurons. The genome-wide view revealed great sequence diversity within the V1r superfamily. Phylogenetic analyses suggested an ancient original radiation, followed by the isolation, divergence and expansion of families by extensive gene duplications and frequent gene loss. The isolated nature of these gene families probably reflects a specialization of different receptor classes in the detection of specific types of chemicals.

Animals↗
PubMed2003

Interisland evolution of Trimeresurus flavoviridis venom phospholipase A(2) isozymes.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A(2) (PLA(2)), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA(2) from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA(2), named PLA-B', which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp(49)]PLA(2) isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B' (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the beta1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA(2), named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA(2) isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA(2) isozymes are clearly separated into three groups, PLA2 type, basic [Asp(49)]PLA(2) type, and [Lys(49)]PLA(2) type. Basic [Asp(49)]PLA(2)-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys(49)]PLA(2) type.

Amino Acid Sequence↗