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Results for “Copy Number Variations”

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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↗

PubMed1995

Adenovirus type 12-induced fragility of the human RNU2 locus requires U2 small nuclear RNA transcriptional regulatory elements.

Infection of human cells with oncogenic adenovirus type 12 (Ad12) induces four specific chromosome fragile sites. Remarkably, three of these sites appear to colocalize with tandem arrays of genes encoding small, abundant, ubiquitously expressed structural RNAs--the RNU1 locus encoding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, and the RN5S locus encoding 5S rRNA. Recently, an artificial tandem array of the natural 5.8-kb U2 repeat unit has been shown to generate a new Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, and S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating that the U2 repeat unit alone is sufficient for virally induced fragility. To identify elements within the U2 repeat unit that are required for virally induced fragility, we generated cell lines containing artificial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragment spanning the U2 gene alone, or the same 834-bp fragment from which key U2 transcriptional regulatory elements had been deleted. The U2 snRNA coding regions within each artificial array were marked by an innocuous single base change (U to C at position 87) so that the relative expression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both the 5.8- and the 0.8-kb U2 repeat units are fragile but that arrays lacking either the distal sequence element or both the distal and the proximal sequence elements of the promoter are not. Surprisingly, variations in repeat copy number and/or transcriptional activity of the artificial arrays do not appear to correlate with the degree of Ad12-inducible fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRNA transcription per se.

Adenovirus Infections, Human↗

PubMed1983

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

Animals↗

PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗

PubMedDate not supplied

A novel MspI PCR-RFLP in the human cytosine 5-methyltransferase gene: lack of relevance for malignant lymphoproliferative disease and breast cancer.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin's lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.

Alleles↗

PubMed1995

Ethnic differences of polymorphism of an immunoglobulin VH3 gene.

The VH26 germline gene occupies two different loci, due to gene duplication, and is one of the most frequently expressed human immunoglobulin VH genes. This report identifies the alleles of each VH26 locus and describes distinct patterns of VH26 polymorphism in three ethnic groups. Oligonucleotide probes targeting VH26 were used in sequence-specific RFLP analysis of DNA from 72 Caucasians, 52 Asians, 35 American Blacks, and members of six families. The A locus, on a 7.0-kb TaqI band, was detected in 89% of Caucasians, 75% of Asians, and 26% of Blacks (chi2 = P < 0.0005). The B locus, detected on a 5.0-kb band in nearly all subjects, was found to have additional alleles occurring at 6.8 kb in 10% of Asians and 3% of Blacks (chi2 = 7.8, P < 0.02) and at 3.7 kb in 1.4% of Caucasians, 21% of Asians, and (9% of Blacks (chi2 = 13.8, P < 0.001). In Asians, only, the 3.7-kb hybridization band represented a multiple-duplication unit containing three or four gene copies. Duplications of other VH26 alleles, and mull alleles of the B locus, were also seen. An exact VH26 sequence was cloned from the 5.0-kb allele and likely exists in the 7.0- and 6.8-kb alleles. A novel sequence cloned from the 3.7-kb allele differed from VH26 by nine nucleotides and appears to have evolved by gene conversion in CDR2. The total diploid gene dose of the A and B loci ranged from one to as many as six copies of VH26-containing genes, and from zero to as many as six to eight copies of the 3.7-kb allele. We conclude that ethnic differences in polymorphism exist at both VH26 loci. These differences could influence VH26 expression because they involve variations in gene copy number and coding region sequence.

Alleles↗

PubMed1995

Characterization of factor VIII gene inversions using a non-radioactive detection method: a survey of 102 unrelated haemophilia A families from northern France.

Intrachromosomal rearrangements of the long arm of chromosome X, between gene A (F8A) in intron 22 of the factor VIII gene and one of its two telomeric copies, are responsible for about half of the severe cases of haemophilia A. A group of 98 unrelated patients from Northern France with moderate to severe haemophilia A was screened for this gene inversion using a non-radioactive Southern blotting method. Whereas none of the 18 moderately affected patients presented the FVIII gene rearrangement, gene inversion was found in 38 (48%) of the 80 severe haemophilia A patients. Recombinations involving the distal copy of gene A (group 1) were more frequent (79%) than those involving the proximal copy (group 2). Individual variation in the number of gene A copies on the X chromosome probably explains an alternative Southern blot profile, referred to as group 3 inversion, which was observed in one of our patients. In the severely affected patients, neither the prevalence of inhibitor development nor the frequency of sporadic cases differed significantly in the group presenting gene inversion as compared to the group without chromosomal rearrangement. Study of four families where no patient was available enabled in one case direct carrier detection and prenatal diagnosis in the absence of an affected member. The Southern blotting technique described in the present work is relevant to about 50% of cases of severe haemophilia A, can be performed without use of a radiolabelled probe and represents a major advance in the diagnosis of the disease.

Biomarkers↗

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↗

PubMed1996

Molecular approaches for the diagnosis of 21-hydroxylase deficiency and congenital adrenal hyperplasia.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in deficiency of cortisol and aldosterone and overproduction of androgens. The 21-hydroxylase locus has a complicated structure, with a highly homologous pseudogene (CYP21P) and an active gene (CYP21) in tandem repeats, a high degree of interindividual variation in gene copy numbers, and exchange of sequences between CYP21P and CYP21. Nine mutations, representing sequences that are normally present in the pseudogene, account for about 95% of all affected CYP21 alleles. Accurate and rapid diagnostic evaluation of congenital adrenal hyperplasia due to 21-hydroxylase deficiency can be performed by typing directly for disease-causing mutations using allele-specific polymerase chain reaction (PCR). A strong correlation exists between genotype and phenotype; mutational analysis can be used to predict disease severity in affected individuals.

Adrenal Hyperplasia, Congenital↗

PubMed2002

The control of natural variation in cytosine methylation in Arabidopsis.

We explore the extent and sources of epigenetic variation in cytosine methylation in natural accessions of the flowering plant, Arabidopsis thaliana, by focusing on the methylation of the major rRNA gene repeats at the two nucleolus organizer regions (NOR). Our findings indicate that natural variation in NOR methylation results from a combination of genetic and epigenetic mechanisms. Genetic variation in rRNA gene copy number and trans-acting modifier loci account for some of the natural variation in NOR methylation. Our results also suggest that divergence and inheritance of epigenetic information, independent of changes in underlying nucleotide sequence, may play an important role in maintaining natural variation in cytosine methylation.

Arabidopsis↗

PubMedDate not supplied

DNMT1 gene copy number does not influence susceptibility to development of malignant lymphoproliferative disease.

BACKGROUND: For many years, the methylating enzyme DNMT1 has been thought to critically influence neoplastic transformation. To determine if DNMT1 is involved in the early events of malignant transformation of lymphoid cells, we investigated whether allele-specific variation in the gene copy number of DNMT1 influences susceptibility to the development of malignant lymphoproliferative disease (LPD) associated with DNA hypermethylation. MATERIALS AND METHODS: DNMT1 gene copy number was assessed by subjecting DNA from DNMT1A/DNMT1B heterozygous patients suffering from benign LPD or malignant LPD featuring Myf-3 hypermethylation to Southern blotting and densitometric analysis. RESULTS: The distribution of DNMT1B to DNMT1A intensity ratios did not significantly differ between patients suffering from benign and patients suffering from malignant LPD and there was no association between increasing Myf-3 hypermethylation and increased DNMT1 gene copy number. CONCLUSION: In contrast to findings in mice, the results of this study exclude a role for DNMT1 gene copy number in susceptibility to neoplastic transformation in humans.

Cell Transformation, Neoplastic↗

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↗

PubMed1992

Genetic variation in the African rodent subfamily Otomyinae (Muridae). II. Chromosomal changes in some populations of Otomys irroratus.

Chromosome-banding studies have been carried out on 31 specimens of Otomys irroratus from six localities. Light-microscope preparations of chromosomes were obtained from cultures of fibroblasts, spleen lymphocytes, peripheral blood lymphocytes, and directly from bone marrow. Karyotypic variability, both numerical and morphological, was detected in three populations. Diploid numbers ranged from 2n = 23 to 2n = 32. Intrapopulation differences were chiefly caused by variation in the number of copies in two pairs of small, biarmed, partly heterochromatic autosomes suggestive of B chromosomes. A major morphological variation in the karyotypes involved the presence of seven pairs of biarmed autosomes with totally heterochromatic short arms in the populations distributed to the west of 26 degrees 57' E. To the east of this longitude, populations of this species exhibited mostly acrocentric autosomes. G-banding patterns of these karyotypes and those of a karyotype from a previous study (Robinson and Elder, 1987) were compared. A chromosome originating from a tandem fusion, possibly leading to partial reproductive isolation, was found in one population. Possible implications of these results for mechanisms of speciation are discussed.

Animals↗

PubMed1994

Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design.

The conditions for the high cell density fed-batch culture of a Saccharomyces cerevisiae strain producing recombinant hirudin (rHV2-Lys47) have been established. A Leu+ derivative of S. cerevisiae c13ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding rHV2-Lys47 and driven by the MF alpha 1 promoter. In order to develop the fed-batch culture protocol, the recombinant strains' physiology was first of all investigated in chemostat culture. The maximum respiratory capacity of the recombinant strain was observed to be between dilution rates of 0.2 and 0.26 h-1, which is typical for laboratory strains as compared to values published for baker's yeasts. Furthermore, maximum biomass yield and product secretion were observed at a dilution rate of approx. 0.15 h-1. The plasmid segregational stability of the recombinant strain showed that the expression plasmid was stable, irrespective of the dilution rates used, for more than 80 generations of growth between dilution rates of 0.043 h-1 and 0.3 h-1. The chemostat data was used to define a fed-batch process. The fed-batch results demonstrated a biomass production of 60 g l-1 CDW and a high production level of recombinant hirudin of 500 mg l-1. Stability of the expression of the gene coding for rHV2-Lys47 was maintained during all the studied fed-batch conditions. The plasmid copy number in the fed-batch remained constant at approx. 43 at a specific growth rate of 0.12 h-1, whereas it increased by 60-95% at a lower dilution rate (mu = 0.06 h-1). Although a variation of the plasmid copy number could be expected, it was postulated from the experimental data that the observed amplification could have been influenced by an environmental effect due to an accumulation of medium components in the supernatant. The results presented here illustrate the importance of a well-balanced medium when considering the production of a recombinant protein in a high cell density cultivation process with high production levels.

Base Sequence↗

PubMed1998

Heteroplasmy and organelle gene dynamics.

This study assesses factors that influence the rates of change of organelle gene diversity and the maintenance of heteroplasmy. Losses of organelle gene diversity within individuals via vegetative segregation during ontogeny are paramount to resultant spatial and temporal patterns. Steady-state losses of organelle variation from the zygote to the gametes are determined by the effective number of organelles, which will be approximately equal to the number of intracellular organelles if random segregation prevails. Both rapid increases in organelle number after zygote formation and reductions at germ lines will reduce variation within individuals. Terminal reductions in organelles must be to very low copy numbers (<5) for substantial losses in variation to occur rapidly. Nonrandom clonal expansion and vegetative segregation during gametogenesis may be effective in reducing genetic variation in gametes. If organelles are uniparentally inherited, the asymptotic expectations for effective numbers of gametes and spatial differentiation will be identical for homoplasmic and heteroplasmic conditions. The rate of attainment of asymptote for heteroplasmic organelles, however, is governed by the rate of loss of variation during ontogeny. With sex-biased dispersal, the effective number of gametes is maximized when the proportional contributions of the sex having the higher dispersal rate are low.

Animals↗

PubMed2001

Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus.

Determination of retroviral load is an important tool in the investigation of the success of therapeutic or vaccination trials in patients infected with lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) counterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detection System (TaqMan) to quantify the viral load of FIV-infected cats. Two different primer/probe systems were designed to detect a broad range of clade A FIV isolates. Both systems are characterized by excellent reproducibility, high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations have greater impact on the accuracy of the viral load estimation. To compensate for these variations, we improved the assay and developed a multiplex real-time RT-PCR, which allows simultaneous calculation of the viral copy number and the individual recovery rate in an one-tube reaction. This enables the rapid and accurate calculation of copy number independent of preassay variations. In further studies, two additional real-time RT-PCR assays were designed and used to investigate the influence of sequence variations in the binding regions for either the primers or probe. Sequence mismatches in this region had a significant effect (up to 4 logarithmic decades) on reaction efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies before determining viral load.

Animals↗

PubMed1992

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Analog-Digital Conversion↗

PubMed1990

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains.

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.

Animals↗