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Results for “DNA Copy Number Variations”

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PubMed2006

Disentangling reasons for low Y chromosome variation in the greater white-toothed shrew (Crocidura russula).

Y chromosome variation is determined by several confounding factors including mutation rate, effective population size, demography, and selection. Disentangling these factors is essential to better understand the evolutionary properties of the Y chromosome. We analyzed genetic variation on the Y chromosome, X chromosome, and mtDNA of the greater white-toothed shrew, a species with low variance in male reproductive success and limited sex-biased dispersal, which enables us to control to some extent for life-history effects. We also compared ancestral (Moroccan) to derived (European) populations to investigate the role of demographic history in determining Y variation. Recent colonization of Europe by a small number of founders (combined with low mutation rates) is largely responsible for low diversity observed on the European Y and X chromosomes compared to mtDNA. After accounting for mutation rate, copy number, and demography, the Y chromosome still displays a deficit in variation relative to the X in both populations. This is possibly influenced by directional selection, but the slightly higher variance in male reproductive success is also likely to play a role, even though the difference is small compared to that in highly polygynous species. This study illustrates that demography and life-history effects should be scrutinized before inferring strong selective pressure as a reason for low diversity on the Y chromosome.

Animals↗

PubMed1993

Chromosomal polymorphism and sexual differentiation in Plasmodium.

The correlation observed in several instances between the loss of ability to produce gametocytes and chromosomal rearrangements, prompted us to investigate in further detail the molecular bases of chromosomal polymorphism in Plasmodium. Generation of polymorphic karyotypes in Plasmodium involves important rearrangements, mostly occurring in subtelomeric position. Detailed analysis on the organisation of these regions have been carried out on the rodent malaria P. berghei and the human malaria P. falciparum. A 2.3kb sequence, tandemly organised in long clusters is shared by many P. berghei chromosomal ends. Variations in the copy number of this "module" account for most of the observed polymorphisms. In a P. falciparum cloned line (3D7) a common region spanning at least 40 kb, is present. It does not contain any repetitive structure other than the rep20 cluster, that appears to be completely contained within the common region. Notwithstanding the structural differences, human and rodent Plasmodia share the common feature of possessing long subtelomeric regions showing, thus, a homology between the different chromosomes.

Animals↗

PubMed2002

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for approximately 60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from approximately 20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.

Animals↗

PubMed2002

Optimization of electroporation conditions for Mycobacterium avium.

Successful transformation and subsequent genetic manipulation of Mycobacterium avium requires suitable vectors, efficient transformation systems, and reliable selectable markers. A systematic analysis of the parameters involved in the transformation of M. avium was performed to optimize DNA transfer. Factors examined included the composition of the growth medium, growth medium additives, variations in washing of the bacteria prior to electroporation, and conditions of electroporation. Of the parameters assayed, the frequency of transformation (defined as the number of transformants per 10(6) transformed bacteria) showed the greatest increase with the addition of 1.5% glycine to the M. avium culture medium and the use of higher concentrations of plasmid DNA. The addition of 0.5 M sucrose to the growth medium and wash solution yielded a modest increase in transformation frequency, but more importantly afforded greater consistency of results between different batches of cells with no decrease in transformation yields following freezing and thawing. We also confirmed that gfp could be used as a selective marker for M. avium, even as a single copy integrant, and allowed for rapid discrimination between false and true transformants. Using this protocol, we were able to transform nine of 11 clinical strains of M. avium.

Culture Media↗

PubMed2004

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene.

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.

Base Sequence↗

PubMed1995

Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians.

To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals.

Adolescent↗

PubMed2005

Diversity and clustered distribution of retrotransposable elements in the compact genome of the pufferfish Tetraodon nigroviridis.

We report the characterization and chromosomal distribution of retroelements in the compact genome of the pufferfish Tetraodon nigroviridis. We have reconstructed partial/complete retroelement sequences, established their phylogenetic relationship to other known eukaryotic retrotransposons, and performed double-color FISH analyses to gain new insights into their patterns of chromosomal distribution. We could identify 43 different reverse transcriptase retrotransposons belonging to the three major known subclasses (14 non-LTR retrotransposons from seven clades, 25 LTR retrotransposons representing the five major known groups, and four Penelope-like elements), and well as two SINEs (non-autonomous retroelements). Such a diversity of retrotransposable elements, which seems to be relatively common in fish but not in mammals, is astonishing in such a compact genome. The total number of retroelements was approximately 3000, roughly representing only 2.6% of the genome of T. nigroviridis. This is much less than in other vertebrate genomes, reflecting the compact nature of the genome of this pufferfish. Major differences in copy number were observed between different clades, indicating differential success in invading and persisting in the genome. Some retroelements displayed evidence of recent activity. Finally, FISH analysis showed that retrotransposable elements preferentially accumulate in specific heterochromatic regions of the genome of T. nigroviridis, revealing a degree of genomic compartmentalization not observed in the human genome.

Animals↗

PubMed2005

Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei.

Arbuscular mycorrhizal fungi (AMF) are ancient asexually reproducing organisms that form symbioses with the majority of plant species, improving plant nutrition and promoting plant diversity. Little is known about the evolution or organization of the genomes of any eukaryotic symbiont or ancient asexual organism. Direct evidence shows that one AMF species is heterokaryotic; that is, containing populations of genetically different nuclei. It has been suggested, however, that the genetic variation passed from generation to generation in AMF is simply due to multiple chromosome sets (that is, high ploidy). Here we show that previously documented genetic variation in Pol-like sequences, which are passed from generation to generation, cannot be due to either high ploidy or repeated gene duplications. Our results provide the clearest evidence so far for substantial genetic differences among nuclei in AMF. We also show that even AMF with a very large nuclear DNA content are haploid. An underlying principle of evolutionary theory is that an individual passes on one or half of its genome to each of its progeny. The coexistence of a population of many genomes in AMF and their transfer to subsequent generations, therefore, has far-reaching consequences for understanding genome evolution.

Cell Nucleus↗

PubMed1978

Evidence for variation in the number of functional gene copies at the AmaR locus in Chinese hamster cell lines.

The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.

Amanitins↗

PubMed1991

Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position.

The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter. Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene. We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome. The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells. Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60%. Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines. The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks. The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks. Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells.

Animals↗

PubMedDate not supplied

Characterization of new PCR based markers for mapping and diagnosis: AC dinucleotide repeat markers at the DXS237 (GMGX9) and DXS102 (cX38.1) loci.

Genomic insert DNAs from 45 probes representing 113.4 kb of the X chromosome were screened for AC dinucleotide repeat sequence. Two new AC repeat sequences were identified with length polymorphism based on variation in repeat copy number. One at DXS237 exhibits 44% heterozygosity and is potentially useful for rapid diagnosis and mapping of X-linked disorders in Xp22.3. The other, at DXS102 in Xq26, has 71% heterozygosity. This marker will improve accuracy of diagnoses by linkage for families with Börjeson-Forssman-Lehmann syndrome. Review of the literature has identified 31 PCR based markers on the X chromosome, with minimum heterozygosity of 50%, applicable to the mapping and diagnosis of X-linked disorders.

Base Sequence↗

PubMed1995

Temperature-gradient gel electrophoresis as a screening tool for polymorphisms in multigene families.

Information about sequence variability between different copies of a multigene family is indispensable for understanding the evolutionary mechanisms acting on multigene families. However, their high copy number has been a major obstacle to systematic analysis. Exemplified by the internal transcribed spacer 1 (ITS1) of the rDNA in Drosophila melanogaster, it is shown how temperature gradient gel electrophoresis (TGGE) can be used to study sequence polymorphisms in a multigene family. Experimental conditions influencing the melting behavior of the ITS1 fragment are discussed as well as discrepancies between observed and calculated melting patterns.

Animals↗

PubMed1979

Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster.

The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.

Animals↗

PubMed1993

Mitotic stability and meiotic variability of the (CAG)n repeat in the Huntington disease gene.

The gene causing Huntington's disease, an autosomal dominantly inherited, neurodegenerative disorder, has been identified recently. The corresponding mutation is involving an expansion in the number of (CAG)n repeats in the coding region of the Huntington's disease gene on chromosome 4. In this report, we demonstrate the length variation of the repeat in 513 non-HD chromosomes from normal individuals and HD patients showing 23 alleles with 11 to 33 repeats. Analyzing the inheritance of the (CAG)n stretch we found meiotic instability for HD alleles ([CAG]40 to [CAG]75) with a mutation frequency of approximately 0.7, while in 431 meioses of normal alleles only two expansions were identified. The risk of expansion during spermatogenesis is enhanced compared to oogenesis explaining juvenile onset by transmission from affected fathers. Further, the number of (CAG)n copies in an affected individual in relation to the sex of the transmitting parent was evaluated and no significant differences were found. No mosaicism or differences in the repeat lengths were observed in the DNA from different tissues including brain and lymphocytes of two HD patients indicating mitotic stability of the mutation. Therefore, the determination of the repeat number in the DNA of blood lymphocytes is probably representative of all tissues in a patient.

Alleles↗

PubMed2004

Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification.

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.

Alleles↗

PubMed2002

Allopolyploidization and evolution of species with reduced floral structures in Lepidium L. (Brassicaceae).

Understanding the pattern of speciation in a group of plants is critical for understanding its morphological evolution. Lepidium is the genus with the largest variation in floral structure in Brassicaceae, a family in which the floral ground plan is remarkably stable. However, flowers in more than half of Lepidium species have reduced stamen numbers, and most of these also have reduced petals. The species with reduced flowers are geographically biased, distributed mostly in the Americas and Australia New Zealand. Previous phylogenetic studies using noncoding regions of chloroplast DNA and rDNA internal transcribed spacer were incongruent in most New World species relationships. These data, combined with the presence of many polyploid Lepidium species, implied a reticulate history of the genus but did not provide enough information to infer the evolutionary pattern of flower structures. To address this question more thoroughly, sequences of the first intron of a single copy nuclear gene, PISTILLATA, were determined from 43 species. Phylogenetic analysis of the PI intron suggests that many species in the New World have originated from allopolyploidization, and that this is correlated with floral reduction. Interspecific hybrids were generated to understand why allopolyploidization is associated with reduced flowers. The phenotypes of F1 flowers indicate allelic dominance of the absence of lateral stamens, suggesting that propagation of dominant alleles through interspecific hybridization could account for the abundance of the allopolyploid species without lateral stamens.

Base Sequence↗

PubMed1992

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.

Animals↗

PubMed1981

Contextural genomic rearrangements of variable-antigen genes in Trypanosoma brucei.

We have described some rearrangements of a variable-antigen gene in T. brucei. We suggest that there are two copies of the ILTat 1.2 variable-antigen gene in each of a number of trypanosome clones closely related by sequential relapses. Both copies of the gene undergo rearrangements, apparently the result of insertion and deletion of lengths of DNA in a region at or beyond the 3' end of the coding sequence, giving rise to different-size restriction enzyme fragments in different clones of trypanosomes. No feature of these rearrangements can be correlated with expression of the gene. Our data differ from those of Hoeijmakers et al. (1980) in two important respects: (1) Neither copy of the gene remains in a constant genomic context in all trypanosome clones. (2) We do not see an extra copy associated with the expression of the gene. These observations do not suggest any obvious mechanism for the phenomenon of antigenic variation.

Animals↗