Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Instability of interstitial telomeric sequences in the human genome

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.     

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.     

Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system

In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids.     

Gene typing of infectious tularemia strains isolated from the Stavropol and Krasnodar Territories

The typing of F. tularensis strains by four variable number of tandem repeats (VNTR) loci has been carried out. Among the strains isolated in the Stavropol and Krasnodar Territories seven genotypes have been detected and their spread in different natural foci has been analyzed. The data thus obtained suggest that the VNTR analysis may become an important instrument for studying the structure of the natural foci of tularemia and evolutionary relationships between individual areas of these foci.     

Mutation in microsatellite repeats of DNA and embryonal death in humans

In the analysis of tetranucleotide DNA repeats inheritance carried out in 55 families with a history of spontaneous miscarriages and normal karyotypes in respect to 21 loci located on seven autosomes, 8 embryos (14.5%) demonstrating 12 cases of the presence of alleles absent in both parents were described. The study of chromosome segregation using other DNA markers permitted highly probable exclusion of false paternity as well as uniparental disomy as the reasons for parent/child allele mismatches. The high probability of paternity together with the presence of a "new" allele at any offspring locus points to the mutation having occurred during game-togenesis in one of the parents. Examination of mutation in spontaneous abortuses revealed an increased number of tandem repeat units at microsatellite loci in three cases and an decreased number of these repeats in six cases. In two abortuses, a third allele absent in both parents, which resulted from a somatic mutation that occurred during embryonic development, was observed. The prevalence of the male germline mutations, revealed during investigation of the mutation origin, was probably associated with an increased number of DNA replication cycles in sperm compared to the oocytes. In spontaneous abortuses, the mean mutation rate of the tetranucleotide repeat complexes analyzed was 9.8 x 10(-3) per locus per gamete per generation. This was about five times higher than the spontaneous mutation rate of these STR loci. It can be suggested that genome instability detected at the level of repeated DNA sequences can involve not only genetically neutral loci but also active genomic regions crucial for embryonic viability. This results in cell death and termination of embryonic development. Our findings indicate that the death of embryos with normal karyotypes in most cases is associated with an increased frequency of germline and somatic microsatellite mutations. The data of the present study also provide a practical tool for the quantitative evaluation of this phenomenon and for the analysis of the reasons for miscarriages and embryonic death in certain families.     

Variable tandem repeat VcA of Vibrio cholerae

Computer analysis revealed seven potential variable-number tandem-repeat (VNTR) loci in the Vibrio cholerae genome. Specific primers were designed to amplify locus VcA located on chromosome 2 and containing a TGCTGT repeat. The locus was found in all tested strains from a V. cholerae strain collection, the repeat number varying 3 to 23. In total, 14 VcA alleles were observed. The VcA locus was proposed as a marker for the molecular typing of V. cholerae strains.     

Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients

Serial sputum isolates of Haemophilus influenzae (n = 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.     

Development of microsatellite markers for blunt snout bream Megalobrama amblycephala using 5'-anchored PCR

A rapid method for isolating microsatellite loci in blunt snout bream, based on the 5'-anchored polymerase chain reaction technique, revealed 522 microsatellite loci (consisting of 442 dinucleotide, 4 trinucleotide and 76 tetranucleotide repeats). Of the 25 loci characterized, 10 turned out to be highly polymorphic. The number of alleles per locus ranged from 3 to 17 while the expected heterozygosity ranged from 0.4899 to 0.9355 in population of selected strain F(7 ) and from 0.5786 to 0.9556 in wild population from Lake Liangzi. These markers are useful as tools for the detection of genetic variation levels in selected strains and wild populations of blunt snout bream for germplasm conservation.     

PCR-based rapid genotyping of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>isolates

Background  

All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA).     

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.     

Human minisatellite alleles detectable only after PCR amplification.

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles funestus

Screening of the Anopheles funestus genomic DNA library detected 18 new sequences with dinucleotide tandem repeats. Primers were designed to amplify the loci and 14 out of 18 gave a repeatable and scorable amplification. Deviations from Hardy–Weinberg expectations were tested for each locus in a sample of 30 wild Anopheles funestus females. No heterozygote deficiency was detected for 11 loci of 14, thus revealing the absence of null alleles. The number of alleles per locus ranged from 5 to 15, and observed heterozygosity from 0.13 to 0.85.     

Chromosome rearrangement and diversification of Francisella tularensis revealed by the type B (OSU18) genome sequence

The gamma-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp. tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp. holarctica sequence with that of F. tularensis subsp. tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).     

DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.     

Differentiation of strains of Xylella fastidiosa by a variable number of tandem repeat analysis

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.