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Results for “DNA Copy Number Variations”

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PubMed2006

Differences in the copy number of the py235 gene family in virulent and avirulent lines of Plasmodium yoelii.

The 235kDa rhoptry protein (Py235) of Plasmodium yoelii is coded for by a multigene family. Py235 has been implicated in host cell selection and virulence as antibodies against it have been shown to inhibit invasion of mature red blood cells of the normally virulent P. yoelii YM line and at least one member of this family directly binds to erythrocytes. Differences in py235 sequence and copy number have been postulated to be responsible for the differences in invasion phenotype seen in the avirulent P. yoelii YA line and the YM line. The newly available sequence data for P. yoelii 17X NL 1.1 has now made it possible to investigate this further. A number of approaches including real time PCR was used to determine the exact copy number of individual py235. Except for two cases in YA and one in YM there are no differences in py235 copy number between the two lines and 17X NL 1.1. Analysis of progeny of a genetic cross between YM and an avirulent strain AC yield similar limited variations in copy number. This study shows that the copy number of py235 in the analyzed P. yoelii strains is significantly lower than previous estimates and much more in line with the published genome sequence. The lower copy number as well as the limited difference of py235 in the virulent lines makes it highly unlikely that these are the factors contributing to the differences in invasion observed.

Animals↗

PubMed1983

An African trypanosome variant surface glycoprotein gene whose expression is not activated by duplication.

A variant surface glycoprotein (VSG) of Trypanosoma brucei is encoded by a gene whose expression is not governed by duplication-transposition. There are two copies of this gene. The 5' flanking regions of the two genes are indistinguishable by restriction mapping, although each possesses approximately 5-10 Kbp of DNA which is devoid of restriction sites. All restriction enzymes tested appeared to cut genomic DNA at a uniform distance 3' of the gene. This, coupled with the observed sensitivity of both genes to BAL 31, indicates that they lie near chromosomal termini. Length variation occurs 3' of these genes in bloodstream clones and their procyclic derivatives, although the number of length variants is conserved. This suggests that length variation alone does not control VSG switching or gene expression and that constraints exist on the extent to which 3' flanking regions can vary in length.

Animals↗

PubMed2005

Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Adult↗

PubMed2005

Allele-specific amplification in cancer revealed by SNP array analysis.

Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.

Alleles↗

PubMed2001

Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans.

The capacity of Shiga toxigenic Escherichia coli (STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of saa cloned in pBC results in a 9.7-fold increase in adherence of E. coli JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of saa in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of saa were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of Yersinia enterocolitica and Eib, a recently described phage-encoded immunoglobulin binding protein from E. coli. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.

Adhesins, Bacterial↗

PubMed2003

Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity.

Molecular tools based on rRNA (rrn) genes are valuable techniques for the study of microbial communities. However, the presence of operon copy number heterogeneity represents a source of systematic error in community analysis. To understand the types and magnitude of such bias, four commonly used rrn-based techniques were used to perform an in silico analysis of a hypothetical community comprised organisms from the Comprehensive Microbial Resource database. Community profiles were generated, and diversity indices were calculated for length heterogeneity PCR, automated ribosomal integenic spacer analysis, denaturing gradient gel electrophoresis, and terminal RFLP (using RsaI, MspI, and HhaI). The results demonstrate that all techniques present a quantitative bias toward organisms with higher copy numbers. In addition, techniques may underestimate diversity by grouping similar ribotypes or overestimate diversity by allowing multiple signals for one organism. The results of this study suggest that caution should be used when interpreting rrn-based community analysis techniques.

DNA, Bacterial↗

PubMed1983

Distribution of insertion element IS1 in natural isolates of Escherichia coli.

Total DNAs from twelve natural isolates of Escherichia coli from animals and humans were examined by hybridization with a probe for IS1. Considerable variation in copy number was found. In the case of two strains isolated from the same individual, one strain contained no copies of IS1 and the other, much greater than 30. Evidence was also obtained for the existence of IS1-like elements (iso-IS1s) of greater than 15% sequence divergence relative to the IS1 from antibiotic resistance plasmid R100.

Animals↗

PubMed2000

Association of estrogen receptor beta gene polymorphism with bone mineral density.

Chromosomal mapping of the human estrogen receptor beta (ERbeta) gene by fluorescence in situ hybridization (FISH) reveals that ERbeta is located at human chromosome 14, region q23-24.1, where the aberration of DNA copy number in the bone disorders is frequently involved. Then, we investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphism located in the flanking region of ERbeta gene and bone mineral density (BMD) in 204 healthy postmenopausal Japanese women. The genotype was classified into "A" through "O" according to the number of the repeats, from 18 to 32. BMD was expressed in Z score (a deviation from the weight-adjusted average BMD of each age using the standard deviation as a unit). When we separate the subjects into two groups bearing at least one I allele (26 CA repeats) and those who did not, the former subjects had significantly higher Z score of L2-4 BMD (mean +/- standard deviation; 0.674 +/- 1.53 vs 0.128 +/- 1.38; P = 0.027). These data suggest that genetic variation at the ERbeta locus may be associated with some determinants for BMD and the possible involvements of this polymorphism in the cause of postmenopausal osteoporosis in Japanese women.

Base Sequence↗

PubMed2005

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci.

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.

Base Sequence↗

PubMed1996

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews.

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.

Animals↗

PubMed1992

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement.

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed.

Alleles↗

PubMed2005

Quantitative change in mitochondrial DNA content in various mouse tissues during aging.

In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.

Aging↗

PubMed1995

Vertical transmission of the retrotransposable elements R1 and R2 during the evolution of the Drosophila melanogaster species subgroup.

R1 and R2 are non-long-terminal repeat retrotransposable elements that insert into specific sequences of insect 28S ribosomal RNA genes. These elements have been extensively described in Drosophila melanogaster. To determine whether these elements have been horizontally or vertically transmitted, we characterized R1 and R2 elements from the seven other members of the melanogaster species subgroup by genomic blotting and nucleotide sequencing. Each species was found to have homogeneous families of R1 and R2 elements with the exception of erecta and orena, which have no R2 elements. The DNA sequences of multiple R1 and R2 copies from each species indicated nucleotide divergence within each species averaged only 0.48% for R1 and 0.35% for R2, well below the level of divergence among the species. Most copies of R1 and R2 (40 of 47) sequenced from the seven species were potentially functional, as indicated by the absence of premature termination codons or translational frameshifts that would destroy the open reading frame of the element. The sequence relationships of both the R1 and R2 elements from the various members of the melanogaster subgroup closely followed that of the species phylogeny, suggesting that R1 and R2 have been stably maintained by vertical transmission since the origin of this species subgroup 17-20 million years ago. The remarkable stability of R1 and R2, compared to what has been suggested for transposable elements that insert at multiple locations in these same species, may be due to their unique specificity for sites in the rRNA gene locus. Under low copy number conditions, when it is essential for any mobile element to transpose, the insertion specificities of R1 and R2 ensure uniform developmentally regulated target sites that can be occupied with little or no detrimental effect on the host.

Animals↗

PubMed2000

Nucleotide sequence analysis of hypervariable junctions of Haemophilus influenzae pilus gene clusters.

Haemophilus influenzae pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili, hifABCDE, are found inserted in genomes either between pmbA and hpt (hif-1) or between purE and pepN (hif-2). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of H. influenzae strains. The junctions of the hif-1 cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast, hif-2 junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes hicA and hicB) with evidence of frequent recombination. Variation at hif-2 junctions seems to be tied to multiple copies of a 23-bp Haemophilus intergenic dyad sequence. The hif-1 cluster appears to have originated in biogroup aegyptius strains from invasion of the hpt-pmbA region by a DNA template containing the hif-2 genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile "pathogenicity island" not associated with a phage, transposon, or insertion element.

Base Sequence↗

PubMed2004

Lineage-specific gene duplication and loss in human and great ape evolution.

Given that gene duplication is a major driving force of evolutionary change and the key mechanism underlying the emergence of new genes and biological processes, this study sought to use a novel genome-wide approach to identify genes that have undergone lineage-specific duplications or contractions among several hominoid lineages. Interspecies cDNA array-based comparative genomic hybridization was used to individually compare copy number variation for 39,711 cDNAs, representing 29,619 human genes, across five hominoid species, including human. We identified 1,005 genes, either as isolated genes or in clusters positionally biased toward rearrangement-prone genomic regions, that produced relative hybridization signals unique to one or more of the hominoid lineages. Measured as a function of the evolutionary age of each lineage, genes showing copy number expansions were most pronounced in human (134) and include a number of genes thought to be involved in the structure and function of the brain. This work represents, to our knowledge, the first genome-wide gene-based survey of gene duplication across hominoid species. The genes identified here likely represent a significant majority of the major gene copy number changes that have occurred over the past 15 million years of human and great ape evolution and are likely to underlie some of the key phenotypic characteristics that distinguish these species.

Animals↗

PubMed1992

The population biology and evolutionary significance of Ty elements in Saccharomyces cerevisiae.

The basic structure and properties of Ty elements are considered with special reference to their role as agents of evolutionary change. Ty elements may generate genetic variation for fitness by their action as mutagens, as well as by providing regions of portable homology for recombination. The mutational spectra generated by Ty1 transposition events may, due to their target specificity and gene regulatory capabilities, possess a higher frequency of adaptively favorable mutations than spectra resulting from other types of mutational processes. Laboratory strains contain between 25-35 elements, and in both these and industrial strains the insertions appear quite stable. In contrast, a wide variation in Ty number is seen in wild isolates, with a lower average number/genome. Factors which may determine Ty copy number in populations include transposition rates (dependent on Ty copy number and mating type), and stabilization of Ty elements in the genome as well as selection for and against Ty insertions in the genome. Although the average effect of Ty transpositions are deleterious, populations initiated with a single clone containing a single Ty element steadily accumulated Ty elements over 1,000 generations. Direct evidence that Ty transposition events can be selectively favored is provided by experiments in which populations containing large amounts of variability for Ty1 copy number were maintained for approximately 100 generations in a homogeneous environment. At their termination, the frequency of clones containing 0 Ty elements had decreased to approximately 0.0, and the populations had became dominated by a small number of clones containing > 0 Ty elements. No such reduction in variability was observed in populations maintained in a structured environment, though changes in Ty number were observed. The implications of genetic (mating type and ploidy) changes and environmental fluctuations for the long-term persistence of Ty elements within the S. cerevisiae species group are discussed.

Biological Evolution↗

PubMed2005

Quantification of vertical transmission of Murine polyoma virus by real-time quantitative PCR.

Pathogenesis studies of viral infections in vivo require sensitive assay methods. A sensitive and specific real-time quantitative PCR (RQ-PCR) assay was developed to detect Murine polyoma virus (MuPyV) DNA sequences. A quantitative assay to measure the single-copy murine wild-type p53 gene was developed to normalize viral gene copies to cell numbers. Both assays were sensitive over a seven-log dynamic range, with a reproducible detection limit of 10 copies per reaction. To determine viral loads and tissue distribution following vertical transmission of MuPyV, pregnant BALB/c mice were inoculated intraperitoneally with virus in late pregnancy. Progeny animals born to infected mothers were followed for 21 days. Viral loads in four tissues (salivary gland, kidney, liver and spleen) were highest at 7 days after birth and dropped to low levels by 14 and 21 days of age, with loads ranging from 5 to 2 million MuPyV copies per 10(3) cells. Significant animal-to-animal variation occurred. Fourteen of 21 (67%) progeny were virus-positive in one or more tissue samples. Transplacental transmission was observed in 6/7 (86%) litters. Infected fetuses per positive litter ranged from 1/7 (14%) to 5/6 (83%) with viral loads ranging from 5 to 25 417 MuPyV copies per 1000 fetal cells. Maternal tissues and blood were frequently highly positive 2 days after inoculation, but viral loads were low by day 14. This study demonstrated the vertical transmission, including transplacental transmission, of MuPyV following acute infection of pregnant mice. It should be considered that there is a possibility that other polyomaviruses, including those in humans, may be vertically transmitted.

Animals↗

PubMed2003

Downregulation of Trypanosoma brucei VSG expression site promoters on circular bacterial artificial chromosomes.

Trypanosoma brucei has about 20 telomeric variant surface glycoprotein (VSG) gene expression sites (ESs), which are downregulated in the insect form. We investigated the transcriptional behaviour of ES promoters on bacterial artificial chromosomes (BACs) containing two different ESs and their flanking regions on fragments of about 140kb. Four different BACs containing either the 221 or the VO2 ES were introduced into insect form T. brucei. The BACs replicated as circular episomes as shown using pulsed field gel (PFG) analysis of DNA exposed to increasing doses of gamma radiation, and digestion with Dam methylation-sensitive restriction enzymes. BAC copy number per cell varied from about 3 for the 221 ES BACs to about 15 for the VO2 ES BACs. Increasing drug selection pressure on the VO2 BAC T. brucei transformants resulted in amplification to about 80 BACs per cell. Although BACs were maintained in the absence of drug selection for at least 56 days, copy number fell and there was no evidence for centromere activity. ES promoters on small plasmid episomes introduced into insect form T. brucei in transient transfections are derepressed. In contrast, ES promoters on large BAC episomes are downregulated both on the original ES BACs, and on ES BACs selected for a drug marker driven by a rDNA promoter fused to the BAC vector. This indicates that downregulation of ES promoters in insect form T. brucei is influenced by genomic context, but does not necessitate proximity to a chromosome end.

Animals↗