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PubMed1988

Intrinsic polymorphism of variable number tandem repeat loci in the human genome.

In the human genome, short tandem repetitive (STR) DNA sequences often show restriction fragment length polymorphisms (RFLPs) due to variation in the number of copies of the repeat unit. For a subset of these sequences known as minisatellites or variable number tandem repeat loci (VNTR), it has been proposed that a homologous "core" sequence of 10-12 nucleotides is involved in the mechanism(s) generating the polymorphism. In our present study we have prepared oligonucleotide probes complementary to one or two repeat units of several VNTR loci. Under stringent hybridization and wash conditions these probes hybridize locus specifically thus allowing the evaluation of the intrinsic polymorphism of individual loci. Our results indicate that not all of the loci having STR DNA sequences are polymorphic despite the fact that they share the "core" sequence. This suggests that more than the DNA sequence of the locus is involved in the mechanism(s) generating the polymorphism.

Alleles↗

PubMed1976

DNA reassociation kinetics in relation to genome size in four amphibian species.

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Triturus cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass. Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount. The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.

Amphibians↗

PubMed1995

Isolation, characterization and RFLP linkage mapping of a DNA repeat family of Solanum spegazzinii by which chromosome ends can be localized on the genetic map of potato.

In a random sample of 2263 cloned genomic DNA fragments of the wild potato species Solanum spegazzinii six related, highly repetitive fragments (SPG repeat family) were identified that were present in much higher copy numbers in S. spegazzinii when compared with the closely related cultivated potato S. tuberosum. The SPG repeat family was organized in long arrays of multiple copies. Cross hybridization experiments with 29 wild and cultivated Solanum species and with the related tomato showed specificity of the SPG repeat family for tuber-bearing Solanum species. Among tuber bearing Solanum species a high degree of variation was observed for restriction fragment length and copy number. The variation in copy number was not correlated with established taxonomic relationships between tuber-bearing Solanum species. DNA sequence analysis revealed a subrepeat structure of 120-140 base pairs embedded in longer repeat units of variable length. Length polymorphisms between highly repeated restriction fragments detected by the SPG probes were used for segregation- and linkage analysis in four mapping populations of potato, for which RFLP maps had been constructed. Twelve loci were identified, eleven of which mapped to the distal ends of nine linkage groups. All the evidence suggested that the SPG repeat family represents a satellite repeat members of which are localized in the subtelomeric region of potato chromosomes. The SPG repeat family could be used, therefore, for completing the genetic map of potato.

Base Sequence↗

PubMedDate not supplied

Hypervariable 'minisatellite' regions in human DNA.

The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.

Alleles↗

PubMed1992

Apparent heterozygote deficiencies observed in DNA typing data and their implications in forensic applications.

Restriction fragment length polymorphisms (RFLP) analysis using the Southern blot technique can be used to recognize copy number variation of variable number of tandem repeats (VNTR) of conserved core sequences at several regions of the human genome. This new class of polymorphisms reveals a high degree of genetic variation, useful for individual identification purposes. Criticisms against forensic applications of such DNA typing data include the limitation of employing Hardy-Weinberg expectation of genotype frequencies, since several surveys indicate apparent deficiency of heterozygosity (or excess homozygosity) in comparison with Hardy-Weinberg expectations. This research postulates an alternative explanation of deficiency of apparent heterozygosity which is caused by the inability to detect extremely small-sized alleles (called 'non-detectable' alleles) due to the sensitivity of Southern gel electrophoresis. We show that the presence of 'non-detectable' alleles can produce pseudo-homozygosity and their frequencies can be predicted from the observed proportional heterozygote deficiency. Furthermore, in the covert presence of such 'non-detectable' alleles, we show that the gene-count method provides over-estimates of allele frequencies in the sample population, and hence the Hardy-Weinberg predictions of genotype frequencies avoid wrongful bias against suspects in forensic applications of DNA typing data. Applications of this theory to population data on six VNTR loci in US Caucasians and US Blacks suggest that the presence of 'non-detectable' alleles could be the major cause of apparent heterozygote deficiency, and the current approaches of predicting the population frequency of specific DNA phenotypes are practically free of the possible wrongful bias in courtroom applications of DNA typing data.

Alleles↗

PubMed1994

Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci.

We have examined the performance and reproducibility of an automated DNA profiling system which is based on the multiplex amplification of 4 tetrameric STR loci-HUMVWFA31/A. HUMTH01, HUMF13A1 and HUMFES/FPS. The system was able to type 100 pg of purified, undegraded, genomic DNA. At lower concentrations of DNA (below 100 pg), allelec drop-out occurred due to stochastic differences in allele copy number. Minor variation of individual PCR reagent concentrations or cycling temperatures did not result in a significant effect on the efficiency of amplification of any of the 4 loci in the quadruplex system. More substantial variation of reagent concentrations or cycling temperatures outside the optimum range of the system resulted in a reduction or complete loss of signal for one or more loci. This was also observed at high ionic strength or extreme pH. However, under all reagent concentrations and conditions studied, no artefact bands that could potentially result in the mistyping of a sample were apparent within the read region (130-240 bases) of the gel. Evaluation of both native and denaturing polyacrylamide gels revealed that, although native gels displayed faster run times, the sizing precision of such gels for certain STR loci was lower than that of denaturing gels. Also, artefact bands may be present within the read region of native gels. In conclusion the quadruplex amplification system described, coupled with automated fluorescence-based detection on denaturing polyacrylamide gels, appeared to be a robust and reliable system for individual identification.

Alleles↗

PubMed1986

Close linkage of human chromosomal pepsinogen A genes.

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.

Cloning, Molecular↗

PubMed1989

[EcoRI- and BamHI-families of repeated sequences in mustelids].

The restriction enzymes EcoRI and BamHI digest the genomic DNAs from six mustelids species Mustela lutreola, M. vision, M. erminea, M. sibirica, Vormela peregusna, producing repeated fragments varying in length. Some fragments were hybridized to chromosomes and restriction digests of DNAs from some mustelids and other mammals. The 0.7 kb EcoRI repeats from DNA of M. erminea are dispersed over chromosomes of carnivors. The 1.35, 1.9 and 2.7 kb BamHI repeats from DNA of polecat M. putorius furo are specific for mustelids. These repeats demonstrate interspecific variation in length and the number of copies. All BamHI repeats have no strict tandem organization. The 1.9 kb BamHI repeats are concentrated in the heterochromatic pericentromeric regions and additional chromosome arms. The 1.35 kb BamHI repeats are only located in the centromeric regions of chromosomes of five species and are absent in Vormela peregusna.

Animals↗

PubMed1981

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum.

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33 258 -- CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 x 10(5) copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a 'theme and variation' pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.

Animals↗

PubMed1987

Epstein-Barr virus genomes in lymphoid cells: activation in mitosis and chromosomal location.

Biotin-labeled Epstein-Barr virus (EBV)-specific DNA probes have been used to detect viral genomes by in situ hybridization. Immunocytochemically amplified signals produced by the hybridized probe allow visualization of viral DNA even in cells previously reported to contain only one or two EBV genomes. In EBV producer lymphoid cell lines, such as B95-8, P3HR-1, or Daudi, activation of latent EBV DNA could be observed in mitotic cells; in non-virus-producing cells of these same lines, EBV was found to be present in low copy numbers. Noninducible cell lines such as IB4, AW-Ramos, and Namalwa exhibited low but clearly positive hybridization. Unexpectedly, significant variations in the amounts of EBV DNA per cell were observed between individual cells of these lines. The EBV DNA in the cloned IB4 cell line was localized to chromosome 4 in metaphase cells, but in the noncloned converted line AW-Ramos, the location of integrated viral DNA was essentially random.

Cell Line↗

PubMed1987

Intraspecific and interspecific variation in the sequence and abundance of highly repeated DNA among mosquitoes of the Aedes albopictus subgroup.

Interspecific variation in abundance of highly repeated DNA sequences has been examined in three species of the Aedes scutellaris and three species of the A. albopictus subgroup of the A. scutellaris group. Sequences from a population of A. albopictus were hybridised with whole genomic content from other species and strains. Copy number estimates were determined by dot-blot hybridisation. Variation in sequence abundance between strains of A. albopictus was as great as between it and the other six species. Two clusters were formed by principal components analysis, each of which contains populations of A. albopictus. Copy numbers of highly repeated sequences do not correlate with genome size. The results indicate extensive sequence divergence and rapid evolution. These findings are discussed in relation to the importance of concerted evolution and natural selection in the evolution of the species group.

Aedes↗

PubMed1986

Short repeats cause heterogeneity at genomic terminus of bovine herpesvirus 1.

Analysis of the genomes of different bovine herpesvirus 1 strains revealed a UL terminal HindIII fragment differing in size (from 2.4 to 2.8 kilobases). This fragment polymorphism occurred in the DNA of a wild-type isolate, in highly passaged, apathogenic tissue culture derivatives, and in plaque-purified substrains. This heterogeneity was due to variations in the copy number of a 14-base-pair tandem repeat comprising the base sequence 5'-GCTCCTCCTCCCTC-3', which also exists, with some differences, in other short reiteration sequences of herpes simplex virus type 1, Epstein-Barr virus, and related human cellular DNA. Furthermore, the tandem repeat array was located in close proximity to the left end of the viral genome and may functionally be involved in viral replication.

Animals↗

PubMed1992

16S rrn gene copy number in Helicobacter pylori and its application to molecular typing.

The copy number of the genes encoding 16S ribosomal RNA was analysed for the genomes of geographically diverse strains of Helicobacter pylori, and restriction site variation within and around the genes was characterized. A DNA probe of 550 bp was amplified by the polymerase chain reaction from genomic DNA of the type strain NCTC 11637. This probe constituted a sequence internal to the 3' end of the 16S rrn gene. Homology profiles were compared for genomic Southern blots made with four restriction enzymes cutting within and outside the probe sequence. A copy number of two was established for all 12 strains analysed. This approach yielded significantly simpler data than does conventional 'ribotyping ' of H. pylori. It was equally discriminatory, however, and provided strain-specific 16S rrn gene 'signatures'. These represent both fundamental physical-genetic information and a novel approach to typing this gastric pathogen.

Bacterial Typing Techniques↗

PubMed2025

MarkerMatch: A Proximity-Based Probe-Matching Algorithm for Joint Analysis of Copy-Number Variants from Different Genotyping Arrays.

MOTIVATION: Copy-number variants (CNVs) are a form of genetic structural variation with increasing importance in complex human disorders. Both DNA sequencing and microarray data can be used to call CNVs, which can be used in association tests, such as association between CNV number and disease status. Unlike genotypes, CNV detection in microarrays requires the use of observed intensity signals at each probe, which limits the imputability for analyses that span multiple array types. Thus far, a consensus set of probes (the intersection encompassing the probes that occur in common on all arrays) has been used to circumvent the problem of differing array-specific sensitivities. This has, however, led to excessive reduction in overall sensitivity of CNV calls as arrays can have an undesirably low overlap of probe sets. To overcome this limitation, we developed MarkerMatch, a proximity-based algorithm that matches probes across different genotyping microarrays to maximize the number of probes considered in the CNV calling algorithm, thereby increasing the resolution and sensitivity while preserving precision. RESULTS: By analyzing CNV calls from 4,906 individuals genotyped across three different arrays (Global Screening Array, Omni2.5 array, and Omni Express Exome array), we show that the MarkerMatch approach improves sensitivity by increasing the density of probes available for CNV calling while maintaining precision or improving it relative to the current practice (e.g., use of consensus probes only). We further demonstrate that MarkerMatch exceeds the output from current practice in terms of F1 score, Fowlkes-Mallows index, and Jaccard index. We also optimize MarkerMatch parameters, D MAX and Method, and find an optimal D MAX setting at 10kb, with no clear optimal candidate based on Method, indicating that parameters for this metric should be determined on a use case basis.

Journal Article↗

PubMed1995

Identification of a mariner element from the tsetse fly, Glossina palpalis palpalis.

In the present study, the polymerase chain reaction was used initially to demonstrate the presence of mariner sequences in seven species/subspecies of tsetse flies. DNA hybridization experiments show mariner sequences to be dispersed within the tsetse genome and that there are large variations in copy numbers among the various taxa. A genomic library was used to isolate and characterize a full-length mariner element from G. p. palpalis. The results indicate that this element is 1257 bp in length, flanked by two 32 bp inverted repeats differing at only one position, and belongs to the mellifera subfamily. The nucleotide sequence that is translated into a reading frame of 337 amino acids requires the introduction of two frame shifts and one stop codon to maximize sequence homology with a mariner element from Drosophila erecta. Based on this evidence, we conclude that the G. p. palpalis mariner element clearly represents a non-functional transposable element and that the protein product is not an active transposase.

Amino Acid Sequence↗

PubMed1987

Variable number of tandem repeat (VNTR) markers for human gene mapping.

A large collection of good genetic markers is needed to map the genes that cause human genetic diseases. Although nearly 400 polymorphic DNA markers for human chromosomes have been described, the majority have only two alleles and are thus uninformative for analysis of genetic linkage in many families. A few known marker systems, however, detect loci that respond to restriction enzyme cleavage by producing a fragment that can have many different lengths. This polymorphism is due to variation in the number of tandem repeats of a short DNA sequence. Because most individuals will be heterozygous at such loci, these markers will provide linkage information in almost all families. Ten oligomeric sequences derived from the tandem repeat regions of the myoglobin gene, the zeta-globin pseudogene, the insulin gene, and the X-gene region of hepatitis B virus, were used to develop a series of single-copy probes. These probes revealed new, highly polymorphic genetic loci whose allele sizes reflected variation in the number of tandem repeats.

Chromosome Mapping↗

PubMed1992

Cell phenotype (CD23)-dependent variation in EBV genome copy numbers within lymphoblastoid cell lines (LCL).

Three Epstein-Barr-virus-transformed lymphoblastoid cell lines (LCL) were analysed on the basis of their CD23 expression. Levels of EBV-DNA were compared in the positive and negative subpopulations. Two lines were further analysed with regard to EBNA, cytoplasmic immunoglobulin (cIg) and lytic (EA/VCA) protein expression. Both subpopulations had a similar MHC class-II transcription, but the CD23- subpopulation had a lower plating efficiency and a lower rate of DNA synthesis. In the B6, NAD50 and 0467.3 cell lines, CD23- cells contained 2 +/- 0.2 - 6.4 +/- 3.0 times less EBV DNA than the corresponding CD23+ population. EBNA was expressed in 81 +/- 4.2% - 93 +/- 3.8% of the CD23+ cells and in 0 - 46 +/- 8.0% of the CD23- cells. No CD23+ cells in B6 or NAD50 contained any EA/VCA, while 19 +/- 2.8% - 24 +/- 4.2% of the CD23- cells were positive for the lytic-cycle-associated antigens. Of the CD23- cells, 70 +/- 8.6% - 86 +/- 6.0% were positive for cytoplasmic immunoglobulin compared to 14.7 +/- 2.7% - 14.9 +/- 1.8% in the corresponding CD23+ population. We have previously shown that only 18% of the cIg-positive cells were EBNA-positive in the B6 line compared to 94% in the cIg- population. This was open to 2 alternative interpretations: loss of EBV genomes from a fraction of the cells with subsequent differentiation to secretory immunoglobulin production, or down-regulation of EBNA expression in differentiating, EBV-genome-positive cells. Our present findings speak for the first alternative, indicating that a certain proportion of the cells may lose their EBV genomes in both long-established and freshly transformed LCLs. This is accompanied by a reduced percentage of EBNA-positive cells, the disappearance of at least one activation marker (CD23) associated with the virally induced blast transformation, and an increased synthesis of cIg.

Antibody Formation↗

PubMed2025

SegMantX: A Novel Tool for Detecting DNA Duplications Uncovers Prevalent Duplications in Plasmids.

Segmental duplications play an important role in genome evolution via their contribution to copy-number variation, gene-family diversification, and the emergence of novel functions. The detection of segmental duplications is challenging due to heterogeneous amelioration of sequence similarity among duplicates, which hinders the reconstruction of continuous sequence alignment. Here we introduce SegMantX, a novel approach for the identification of diverged segmental duplications in prokaryote genomes using local alignment chaining. In this approach, local alignments resulting from a preliminary sequence similarity search (e.g. BLASTn) are chained into continuous segments. Evaluating the performance of SegMantX using simulated sequences shows that the tool can detect diverged duplications beyond the sensitivity limits of standard alignment-based methods. Applying SegMantX to 6,784 enterobacterial plasmids, we find that 65% plasmids contain duplicated regions and gene duplications, most of which correspond either to dispersed, noncoding regions or duplicated mobile genetic elements (MGEs; e.g. transposons and insertion sequences). Furthermore, we demonstrate the applicability of SegMantX for the identification of diverged gene transfers between replicons and plasmid hybridization events. Our findings highlight MGEs as drivers of segmental duplications in plasmid evolution, leading to the amplification of their cargo genes, including antibiotic resistance genes. SegMantX provides a powerful framework for reconstructing diverged segmental duplications and other alignment problems.

Plasmids↗