Biomedical subjects

A M Klevytska

Publications and source records attributed to A M Klevytska.

5 recordsLinked to original sources

PubMed2001

Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome.

Yersinia pestis, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the Y. pestis genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the Y. pestis genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (R = 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.

Animals↗

PubMed2000

Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis.

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Bacillus anthracis↗

PubMed2000

vrrB, a hypervariable open reading frame in Bacillus anthracis.

Bacillus anthracis appears to be the most molecularly homogeneous bacterial species known. Extensive surveys of worldwide isolates have revealed vanishingly small amounts of genomic variation. The biological importance of the resting-stage spore may lead to very low evolutionary rates and, perhaps, to the lack of potentially adaptive genetic variation. In contrast to the overall homogeneity, some gene coding regions contain hypervariability that is translated into protein variation. During marker analysis of diverse strains, we have discovered a novel ca. 750-nucleotide open reading frame (ORF) that contains in-frame, variable-number tandem-repeat sequences. Four distinct variable regions exist within vrrB, giving rise to 11 distinct alleles in eight different length categories among B. anthracis strains. This ORF putatively codes for a 241- to 265-amino-acid protein, rich in glutamine (13.2%), glycine (23.4%), and histidine (23.0%). The variable-region amino acids of the vrrB ORF are strongly hydrophilic. Coupled with putative transmembrane domains flanking the variable regions, this suggests a membrane-anchored cytosolic or extracellular location for the putative protein. Sequence analysis of the complete ORFs from three Bacillus cereus strains shows maintenance of the ORF across species boundaries, including strong conservation of the amino acid sequence and the capacity to vary among strains. The presence of 11 different alleles of the vrrB locus is in stark contrast to the near homogeneity of B. anthracis. Evolution of hypervariable genes can negate the lack of genetic variability in species such as B. anthracis and provide select rapid evolution in other more variable species.

Amino Acid Sequence↗

PubMed2000

Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

Alleles↗

PubMed1999

Molecular diversity in Bacillus anthracis.

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.

Animals↗