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PubMed2003

High diversity of ankA sequences of Anaplasma phagocytophilum among Ixodes ricinus ticks in Germany.

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

Anaplasma phagocytophilum↗
PubMed2003

Characterization of Spanish grapevine cultivar diversity using sequence-tagged microsatellite site markers.

A broad germplasm bank collection containing most of the autochthonous Spanish grapevine cultivars was analyzed using six sequence-tagged microsatellite site (STMS) loci: VVS2, VVMD5, VVMD7, ssrVrZAG47, ssrVrZAG62, and ssrVrZAG79. The number of alleles obtained ranged from 9 in ssrVrZAG47 to 13 in VVS2, and the observed genotypes per locus varied between 24 (ssrVrZAG47) and 41 (VVSS2). A total of 57 unique genotypes were obtained considering all 6 loci, and 40 varieties presented at least 1 of these specific genotypes. The genotypic combinations for the 6 loci have generated 163 different profiles in the 176 cultivars. Ten pairs of accessions and one group of four Garnacha's cultivars can not be differentiated. The observed heterozygosity varied between 75.6 (VVMD7) and 90.9% (VVMD5), without significant differences from the expected values for any loci. The VVMD5 locus was the most informative, and also showed the highest discrimination power. The cumulative discrimination power for all six loci was practically 1; however, in fact, these STMS loci have differentiated only about 93% of the accessions, probably owing to high relatedness of the plant material. Usefulness of this STMS set for characterization of a Spanish grapevine collection is emphasized, as well as the elaboration of databases with these molecular markers.

Gene Frequency↗
PubMed1997

[Diversity of DNA sequences among enterohemorrhagic Escherichia coli O157:H7 detected by PCR-based DNA fingerprinting].

A random amplified polymorphic DNA(RAPD) fingerprinting method has been developed to differentiate Enterohemorrhagic Escherichia coli(EHEC) O157:H7 isolates. This method uses an oligonucleotide of arbitrarily chosen sequence to prime DNA synthesis from pairs of sites to which it is matched or practically matched and results in strain-specific arrays of DNA products. By the RAPD analysis using A07(5'-TGCCTCGCACCA-3'), EHEC strains tested in this study were found to be divided into 5 groups. The RAPD arrays among 5EHEC clinical isolates from a single outbreak were identical from each other, and different from other origin. The data indicate that the RAPD method is feasible for investigating the source of an outbreak associated with EHEC O157:H7 infection.

DNA Fingerprinting↗
PubMed1997

Geographic distribution and genetic variability of hepatitis delta virus genotype I.

Three genotypes of hepatitis delta virus (HDV) have been identified, each with different geographic distributions and disease associations. To better define the geographic distribution and genetic variability of HDV genotype I, and to evaluate the extent of genome variability in populations with different patterns of HDV infection, we have analyzed the sequence of HDV RNA in the sera of 72 patients from different areas. Patients were primarily residents of the United States and areas in and around Greece, including Archangelos, Rhodes. All sequences obtained belonged to HDV genotype I, confirming the wide geographic distribution of this genotype and its predominance in Europe and the United States. In contrast to previous studies, phylogenetic analysis of this large and diverse group of sequences, along with all available previously published HDV sequences, showed no well-defined subtypes within genotype I. Low sequence diversity was found for isolates from the United States, Archangelos, Turkey, and Albania, suggesting that HDV was introduced more recently and/or from fewer sources into these areas as compared to mainland Greece, Italy, and north Africa, where sequence diversity is much greater. The low sequence diversity among isolates from Archangelos is particularly interesting in light of the unusually mild pattern of HDV disease found in this community. Comparison of nucleic acid and amino acid sequences within and among genotypes indicated both highly conserved regions as well as genotype-specific sequences that could be related to functional differences. The most distinctive of the latter was that corresponding to the C-terminal 19-20 amino acids of the long form of hepatitis delta antigen, which is highly conserved within each genotype but considerably diverged among them.

Amino Acid Sequence↗
PubMed1996

Characterization of a human alphoid satellite DNA sequence: potential use in assessing genetic diversity in Indian populations.

Sequence analysis of a human repetitive DNA sequence (pTRF5.6) revealed considerable homology (76%) to the alphoid consensus sequence. Genomic blots of StuI-digested human DNA, hybridized to pTRF5.6, generated a ladder of bands with each band corresponding to oligodeoxyribonucleotide of an approx. 170-bp repeat, indicating a tandemly arrayed organization of this repeat element within the genome. Genomic hybridization analyses of unrelated individuals belonging to various geographical regions of India, using this alphoid satellite prove, revealed polymorphic bands ranging between 2 and 9 kb. Along with an individual-specific band pattern, several isomorphic bands below 2 kb were also evident. There was very little genetic variability between populations, suggestive of low polymorphism at the inter-population level. Our result suggest that alphoid satellite sequence probe can be used in assessing the genetic diversity of various ethnic groups/populations belonging to different geographical regions.

Base Sequence↗
PubMed1993

Selection of new biologically active molecules from random nucleotide sequences.

Genetic diversity can be achieved in vitro by inserting random nucleotide (nt) sequences into cloned genes. In the case of enzymes, subsequent genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activities, or altered temperature sensitivities. Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of enzyme. Selecting biologically active DNA sequences from large random populations provides a new method for identifying nt sequences with unique functions. Analogous random sequence selection techniques have been applied to determine the consensus sequence of the Escherichia coli promoters, DNA and RNA sequences that bind specific protein(s), DNA regulatory sequences, ribozyme(s) and ligand-specific RNA(s). In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences into the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have altered substrate specificity and/or catalytic rates when compared to those of the wild-type enzyme.

Base Sequence↗
PubMed1991

Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes.

Nucleic acid sequences of the second exons of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.

Alleles↗
PubMed1991

Nucleotide sequence analysis of isolates of human T-lymphotropic virus type 1 of diverse geographical origins.

Nucleotide sequences for long terminal repeat (LTR), gag, the protease gene, and pol of a human T-lymphotropic virus type 1 (HTLV-1) isolate of probable Caribbean origin (HTLV-1CH) and a Zairian isolate (HTLV-1EL) were determined providing complete proviral sequences for these isolates. These sequences were compared with those available from previously analyzed isolates. Nucleotide sequence differences of 1.2-3.3% were identified among isolates for which complete genetic information was available. Nucleotide sequence diversity was distributed relatively evenly over the genome with 1.3-5.2% differences in the LTR, 1.1-2.9% differences in gag, 0.7-2.1% differences in the protease gene, 0.9-2.5% differences in pol, 0.9-2.4% differences in env, 0.0-1.4% differences in rex, and 0.1-2.6% differences in tax. There were 1.2-2.3% amino acid differences overall, with 0.8-1.6% nonconservative amino acid alterations. Nucleotide differences were not found in regions of the LTR which are important for transcriptional activity or Tax response. Within the Rex-response element, nucleotide differences were found predominantly in loop rather than stem structures, thus, maintaining the overall secondary structure necessary for Rex activity. Evolutionary tree analysis of the sequence differences suggests a predominant clustering of different HTLV1 strains according to geographical origin. An open reading frame was also identified on the minus DNA strand situated between the env and rex/tax genes which exhibits 0.1-6.9% nucleotide sequence variation among HTLV1 strains. The limited sequence variation among HTLV-1 strains is in striking contrast to the extensive heterogeneity seen among human immunodeficiency virus (HIV) strains.

Amino Acid Sequence↗
PubMed2005

Developmental progression of immunoglobulin heavy chain diversity in sheep.

In order to assess the respective impacts of combinatorial rearrangement, junctional diversification, somatic hypermutation and gene conversion in the generation of immunoglobulin heavy chain variable regions diversity, the sequences of 42 variable regions from late fetal, newborn and young sheep were determined and compared to those of adult animals. At earlier stages of development, the use of germline diversity segments appears restricted, junctional variability is already established, and somatic hypermutations are scarce. The sequence diversity in adults is much higher, which we suggest results from a higher hymermutation activity and possibly from the use of a variety of diversity segments. Altogether, this pattern is very reminiscent of the situation observed in cattle, except for the length of the third complementarity determining regions (CDR3) which are shorter in sheep than in bovine. Unlike the chicken and rabbit systems, it seems that new rearrangements continue to occur in sheep for at least several months after birth.

Amino Acid Sequence↗
PubMed2003

Nitrogenase gene diversity and microbial community structure: a cross-system comparison.

Biological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH, has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is non-random and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment.

Archaea↗
PubMed1995

Extensive diversity in repeat unit sequences of the cDNA encoding the polyprotein antigen/allergen from the bovine lungworm Dictyocaulus viviparus.

The complete sequence of the cDNA encoding the nematode polyprotein allergen/antigen (NPA) of the bovine lungworm Dictyocaulus viviparus was obtained by immunoscreening of cDNA expression libraries and by 5' RACE (rapid amplification of cDNA ends). The encoded polypeptide is similar in sequence to the ABA-1 allergen of Ascaris, the gp15/400 'ladder' protein of Brugia malayi, Brugia pahangi and Wuchereria bancrofti, and a 15-kDa antigen of Dirofilaria immitis. As with these, the predicted amino-acid sequence comprises a head-to-tail array of similar polypeptides with regularly spaced consensus proteinase cleavage sites. The D. viviparus protein was designated DvA-1 (D. viviparus antigen-1) and the gene dva-1. The deduced amino-acid sequence of DvA-1 showed features not observed before in other NPAs: (i) a hydrophobic leader peptide is present, (ii) none of the 12 units in the array are identical and the sequences diverge to a degree hitherto unseen in the NPAs of other nematode parasites, (iii) the predicted proteinase cleavage sites are also diverse in sequence and, in two instances, no consensus cleavage site was identifiable at the expected position, (iv) a short repeat unit is present, which is the only one containing a consensus N-glycosylation site and (v) a C-terminal extension peptide is encoded which shows no similarity to that from A. suum ABA-1. Comparison of independent cDNAs revealed slight variations in the sequence of the gene within the parasite population. Antisera to recombinant DvA-1 polypeptide identified 14-15-kDa antigens in both parasite somatic and excretory-secretory material. DvA-1 is the only NPA for which the complete coding sequence is available and the new principles which it illustrates may lie unsuspected in the NPA-encoding genes of all nematode parasites.

Amino Acid Sequence↗
PubMed1995

Junctional region of the myelin basic protein-specific T cell receptor beta chain in mice.

Evidence for the existence of both conserved and diverse amino acid sequences in the junctional regions of the myelin basic protein (MBP)-specific T cell receptors (TCR) in mice is presented. The junctional region of the Nac1-11 MBP peptide-specific, H-2u-restricted TCR beta-chains is characterized by the utilization of similar amino acid sequences. In contrast, diverse junctional sequences within the TCR beta-chains of the p89-101 MBP peptide and H-2s-restricted T cell clones are reported. These findings demonstrate that a limited heterogeneity of the MBP-specific T cell clones does exist. However, it may not be universal even in inbred mouse strains.

Amino Acid Sequence↗
PubMed1998

Sequence-specific polypeptoids: a diverse family of heteropolymers with stable secondary structure.

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide alpha-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the alpha-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

Circular Dichroism↗
PubMed2002

Single-nucleotide polymorphism characterization in species with limited available sequence information: high nucleotide diversity revealed in the avian genome.

As a case study for single-nucleotide polymorphism (SNP) identification in species for which little or no sequence information is available, we investigated several approaches to identifying SNPs in two passerine bird species: pied and collared flycatchers (Ficedula hypoleuca and F. albicollis). All approaches were successful in identifying sequence polymorphism and over 50 candidate SNPs per species were identified from approximately 9.1 kb of sequence. In addition, 17 sites were identified in which the frequency of alternative bases differed by > 50% between species (termed interspecific SNPs). Interestingly, polymorphism of microsatellite/intron loci in the source species appeared to be a positive predictor of nucleotide diversity in homologous flycatcher sequences. The overall nucleotide diversity of flycatchers was 2.3-2.7 x 10(-3), which is approximately 3-6 times higher than observed in recent studies of human SNPs. Higher nucleotide diversity in the avian genome could be due to the relatively older age of flycatcher populations, compared with humans, and/or a higher long-term effective population size.

Animals↗
PubMed2004

Population structure of pathogenic bacteria revisited.

This minireview summarizes the historical development of bacterial population genetic concepts since the early 1980s. Initially multilocus enzyme electrophoresis was used to determine population structures but this technique is poorly portable between laboratories and was replaced in 1998 by multilocus sequence typing. Diverse population structures exist in different bacterial species. Two distinctive structures are described in greater detail. "Young" organisms, such as Yersinia pestis, have evolved or undergone a severe bottleneck in recent millennia and have not yet accumulated much sequence diversity. "genoclouds" in subgroup III Neisseria meningitidis arise because of the accumulation of diversity due to herd immunity, which is then purified during subsequent epidemic spread.

Bacteria↗
PubMed1980

Evidence for a complex class of nonadenylated mRNA in Drosophila.

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 X 10(6) nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 X 10(7) nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that boserved for poly(A+) mRNA, and indicates that congruent to 69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Futhermore, if the number of different structural genes represented in total polysomal RNA is congruent to 1.7 X 10(4), then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numbeology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome.

Animals↗