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Results for “DNA Copy Number Variations”

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PubMed2000

Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome.

Mycobacterial interspersed repetitive units (MIRUs) are 40-100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite-like structures evolve slowly in mycobacterial populations.

Base Sequence↗

PubMed2026

The genetic control of rapid genome content divergence in Arabidopsis thaliana.

Genome evolution in eukaryotes is predominantly driven by the dynamics of repetitive sequences, which vary widely in both copy number and sequence composition. Rates of repeat evolution differ between and within species and are likely modulated by both genetics and environment. To uncover factors shaping the rate of genome content evolution, we analyzed 1,142 resequenced Arabidopsis thaliana genomes using a novel K-mer based approach to characterize genome content variation and identify hypervariable regions underlying differences in repeat abundance. We next treated repeat abundance as a quantitative trait and performed genome-wide association analyses across more than 400 repeat families to identify the genetic basis of copy number variation. Integrating these results through a meta-GWAS approach revealed both cis-acting variants and more than 50 trans-acting loci that regulate repeat abundance genome-wide. Cis-acting variation was predominantly localized to pericentromeric and centromeric regions, whereas trans-acting loci were enriched for candidate genes involved in DNA replication, DNA repair, DNA methylation regulation. Finally, we found evidence that purifying selection acts against mutations that accelerate genome content divergence, favoring alleles that constrain repeat expansion. Together, these findings provide new insights into the genetic architecture and evolutionary forces shaping genome evolution in A. thaliana and establish a framework for investigating these processes in other plant species.

Journal Article↗

PubMed1991

Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli.

Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.

Amino Acid Sequence↗

PubMed2006

Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder.

OBJECTIVES: To determine whether variation in the serotonin transporter gene promoter (5HTTLPR) influences the efficacy of selective serotonin reuptake inhibitors (SSRIs) in generalized social anxiety disorder (GSAD). METHODS: Consecutive series of N=32 patients with DSM-IV GSAD for whom DNA and standardized outcome data from a 12-week SSRI trial were available. After ensuring that neither clinical response [clinical global impression of change scale (CGI-C)] nor 5HTTLPR genotype was confounded by ethnicity or sex, we determined whether the number of copies (0, 1, or 2) of hi-risk alleles using either the diallelic L-S system or the triallelic La-Lg-S system, predicted response and change in Liebowitz social anxiety scale (LSAS) and brief social phobia scale (BSPS) scores during SSRI treatment. RESULTS: Twenty-one patients (66%) were responders to SSRI (i.e., CGI-C much or very much improved). A trend was seen for a linear association between 5HTTLPR genotype and likelihood of response to SSRI: diallelic classification L/L 7/8 (88%), L/S 12/18 (67%), S/S 2/6 (33%), p=0.051; triallelic classification L'/L' 4/5 (80%), L'/S' 14/19 (74%), S'/S' 3/8 (38%), p=0.093. Reduction in LSAS (and BSPS) scores during SSRI treatment was significantly (p<0.02) associated with 5HTTLPR genotype using either the diallelic or triallelic classification. CONCLUSIONS: Variation in a functional polymorphism known to influence serotonin reuptake is associated with SSRI response in patients with GSAD. Independent replication in larger samples is required before the predictive utility of this information can be confirmed and generalized to clinical settings.

Adult↗

PubMed1997

Characteristics and distribution of large tandem duplications in brook stickleback (Culaea inconstans) mitochondrial DNA.

Most animal mitochondrial DNAs (mtDNAs) range in size from 15 to 18 kb, but increased sizes up to approximately 40 kb are occasionally found. We investigated large size variation in mtDNA of the brook stickleback fish, Culaea inconstans, and characterized four large (2.7-5.8 kb) tandem duplications. Duplications differ in size, frequency of occurrence, and degree of associated heteroplasmy, but each includes the control region and one or more adjacent genes. Duplications are correlated with two mtDNA lineages sampled from 31 populations. L1 duplications (3.2-4.8 kb) were present in all lineage I individuals (n = 121, 19 populations); 53 fish were heteroplasmic due to variation in the copy number of a tandemly repeated 270-bp sequence within the duplicated region. In contrast, duplications, L2, L3, and L4 (2.7-5.8 kb) occurred in only 117 of 174 lineage II fish, in eight of 14 populations. Nine fish with L3 or L4 duplications were heteroplasmic, possessing some mtDNAs that lacked duplications (normal-length mtDNAs). Heteroplasmy in L2 was associated with a small variable region near the ND5 gene. Phylogenetic analysis of restriction sites in Culaea mtDNAs and haplotype-defining sequence differences present in both copies argue for multiple independent events that gave rise to three of the four duplications.

Animals↗

PubMed2005

Generating single-copy nuclear gene data for a recent adaptive radiation.

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.

3' Untranslated Regions↗

PubMed1984

Distribution of DNA insertion element IS5 in natural isolates of Escherichia coli.

DNA from Escherichia coli strains in a reference collection of 72 recent natural isolates (ECOR strains) and 25 natural isolates from the "pre-antibiotic" period 1930-1940 (Murray strains) were studied to determine the genomic abundance of insertion element IS5 and the size of genomic restriction fragments carrying sequences homologous to IS5. Among the ECOR strains, nearly two-thirds lack DNA sequences that hybridize with IS5, and one-half of the remainder have only one copy. Among strains in which IS5 is present, extensive variation in the size of IS5-bearing restriction fragments occurs, in many cases allowing distinction among strains that are judged to be nearly identical in genotype because of the identical electrophoretic mobility of the enzyme coded by each of 11 chromosomal loci. Among the Murray strains in which IS5 is present, the average number of elements per strain is larger, but not markedly so, than among recent isolates. Comparison between duplicate strains in the Murray collection suggests that the rate of accumulation of IS5 elements in prolonged storage in stab tubes corresponds to an apparent probability of transposition of approximately 0.008 +/- 0.002 per IS5 element per year. Because of the extensive genetic variation among strains, insertion elements such as IS5 would seem to be convenient genetic markers with which to detect recent common ancestry among strains.

Animals↗

PubMed1988

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.

Adrenal Hyperplasia, Congenital↗

PubMed1999

The plasticity of human telomeres demonstrated by a hypervariable telomere repeat array that is located on some copies of 16p and 16q.

Human telomeres are composed of tandem arrays of TTAGGG repeats with many variant repeats at the proximal ends. Comparison of the interspersion of variant and TTAGGG repeats between alleles can be used to study telomere instability, but the difficulty in identifying chromosome-specific sequences close to the start of autosomal telomeres has hampered such investigations. A chromosome end, including a telomere and adjacent sequence, that is polymorphic for its presence or absence in unrelated individuals has been identified. The telomere-adjacent DNA shows strong homology (92-99%) to sequences, including two expressed sequence tags, that are usually located in subterminal regions of human chromosomes but not adjacent to telomeres. Since this chromosome end arose, it has relocated at least once. In Caucasians, it forms the telomere of approximately 6% of 16q and 2% of 16p chromosome arms. The mechanism of relocation is unknown but must have involved the telomere-adjacent DNA rather than the telomere itself, as copies on 16p and 16q share the same telomere-adjacent sequence. The interspersion patterns of TTAGGG with TGAGGG, TTGGGG and non-amplifying repeat sequences revealed extensive allelic variation, such that 47 different alleles were observed among the 50 alleles mapped. Closely related alleles differ by small changes in copy number at blocks of adjacent like repeats, as seen at the Xp/Yp pseudoautosomal telomere. Such differences are compatible with a model in which the majority of mutations arise by intra-allelic mechanisms, in individuals hemizygous for a single copy of the chromosome end.

Base Sequence↗

PubMed2005

Large-scale structure of genomic methylation patterns.

The mammalian genome depends on patterns of methylated cytosines for normal function, but the relationship between genomic methylation patterns and the underlying sequence is unclear. We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. The genome was found to consist of short (<4 kb) unmethylated domains embedded in a matrix of long methylated domains. Unmethylated domains were enriched in promoters, CpG islands, and first exons, while methylated domains comprised interspersed and tandem-repeated sequences, exons other than first exons, and non-annotated single-copy sequences that are depleted in the CpG dinucleotide. The enrichment of regulatory sequences in the relatively small unmethylated compartment suggests that cytosine methylation constrains the effective size of the genome through the selective exposure of regulatory sequences. This buffers regulatory networks against changes in total genome size and provides an explanation for the C value paradox, which concerns the wide variations in genome size that scale independently of gene number. This suggestion is compatible with the finding that cytosine methylation is universal among large-genome eukaryotes, while many eukaryotes with genome sizes <5 x 10(8) bp do not methylate their DNA.

Animals↗

PubMed1990

The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Animals↗

PubMed1996

Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309).

Many tandemly repeated minisatellite loci display extreme levels of length variation as a consequence of high rates of spontaneous germline mutation altering repeat copy number. Direct screening for new allele lengths by small-pool PCR has shown that instability at the human minisatellite locus MS205 (D16S309) is largely germline specific and usually results in the gain or loss of just a few repeat units. Structural analysis of the order of variant repeats has shown that these events occur preferentially at one end of the tandem array and can result in complex rearrangements including the inter-allelic transfer of repeat units. In contrast, putative mutants recovered from somatic DNA occur at a substantially lower rate and are simple and non-polar in nature. Germline mutation rates vary considerably between alleles, consistent with regulation occurring in cis. Although examination of DNA sequence polymorphisms immediately flanking the minisatellite reveals no definitive associations with germline mutation rate variation, differences in rate may be paralleled by changes in mutation spectrum. These findings help to explain the diversity of MS205 allele structures in modern humans and suggest a common mutation pathway with some other minisatellites.

Alleles↗

PubMed1999

Tsessebe, Topi and Tiang: three distinct Tc1-like transposable elements in the malaria vector, Anopheles gambiae.

Three distinct types of Tc1-family transposable elements have been identified in the malaria vector, Anopheles gambiae. These three elements, named Tsessebe, Topi and Tiang, have the potential to encode transposases that retain most of the conserved amino acids that are characteristic of this transposon family. However, all three are diverged from each other by more than 50% at the nucleotide level. Full-length genomic clones of two types, Topi and Tsessebe, have been isolated and fully sequenced. The third, Tiang, is represented only by a 270 bp, PCR-amplified fragment of the transposase coding region. The Topi and Tsessebe elements are 1.4 kb and 2.0 kb in length, respectively, and differ in the length of their inverted terminal repeats (ITRs). The Topi elements have 26 bp ITRs, whereas the Tsessebe clones have long ITRs ranging in length from 105 to 209 bp, with the consensus being about 180 bp. This difference is due primarily to variation in the length of an internal stretch of GT repeats. The copy number and location of these elements in ovarian nurse cell polytene chromosomes varies greatly between element subtypes: Topi elements are found at between 17-31 sites, Tsessebe at 9-13 and Tiang at 20 euchromatic sites, in addition to several copies of these elements in heterochromatic DNA. The copy number and genomic insertion sites of these transposons varies between A. gambiae strains and between member species of the A. gambiae complex. This may be indicative of transpositionally active Tc1-like elements within the genome.

Amino Acid Sequence↗

PubMed1992

The relapsing fever agent Borrelia hermsii has multiple copies of its chromosome and linear plasmids.

Borrelia hermsii, a spirochete which causes relapsing fever in humans and other mammals, eludes the immune response by antigenic variation of the "Vmp" proteins. This occurs by replacement of an expressed vmp gene with a copy of a silent vmp gene. Silent and expressed vmp genes are located on separate linear plasmids. To further characterize vmp recombination, copy numbers were determined for two linear plasmids and for the 1-megabase chromosome by comparing hybridization of probes to native DNA with hybridization to recombinant plasmids containing borrelial DNA. Plasmid copy numbers were also estimated by ethidium bromide fluorescence. Total cellular DNA content was determined by spectrophotometry. For borrelias grown in mice, copy numbers and 95% confidence intervals were 14 (12-17) for an expression plasmid, 8 (7-9) for a silent plasmid, and 16 (13-18) for the chromosome. Borrelias grown in broth medium had one-fourth to one-half this number of plasmids and chromosomes. Staining of cells with 4',6-diamidino-2-phenylindole revealed DNA to be distributed throughout most of the spirochete's length. These findings indicate that borrelias organize their total cellular DNA into several complete genomes and that cells undergoing serotype switches do one or more of the following: (1) coexpress Vmps from switched and unswitched expression plasmids for at least three to five generations, (2) suppress transcription from some expression plasmid copies, or (3) partition expression plasmids nonrandomly. The lower copy number of the silent plasmid indicates that nonreciprocal Vmp gene recombination may result from loss of recombinant silent plasmids by segregation.

Bacterial Outer Membrane Proteins↗

PubMed1991

Minisatellite-like repeat sequences in the genome of rodent malaria parasites.

Using minisatellite DNA probes and various Southern blots containing DNA samples from rodent malaria parasites it was shown that minisatellite-like sequences occur in the genome of these Plasmodium species. In contrast to the high copy number as observed in higher eukaryotes, the use of fingerprinting techniques on DNA from these parasites reveals that minisatellite sequences are only present at a small number of loci. When parasite lines which differ in biological parameters are compared, no frequent restriction fragment length polymorphism (RFLP) is observed. Screening data banks revealed the presence of repeated copies of one of the probes, which has the monomer sequence CAGGTGG, in the DNA encoding the immunodominant peptide repeat region of the circumsporozoite (CS) protein from a Plasmodium cynomolgi strain. Comparing the sequences for the CS protein from a number of strains of P. cynomolgi revealed that the core region of the repeats, though subject to limited variation, shares homology to the bacterial recombination signal sequence Chi (GCTGGTGG). The implications of the above findings for genetic variation in malaria parasites and evolution of minisatellite sequences will be discussed.

Animals↗

PubMed1998

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy.

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription-PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500-15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Agrobacterium-mediated transformation of Bangladeshi Indica rices.

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.

Agrobacterium tumefaciens↗

PubMed1986

Interstrain variation in amylase gene copy number and mRNA abundance in three mouse tissues.

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.

Animals↗