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Results for “Copy Number Variations”

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PubMed1995

Variable numbers of simple tandem repeats make birds of the order ciconiiformes heteroplasmic in their mitochondrial genomes.

We have analyzed a variable domain of the mitochondrial DNA control region of 18 avian species. Intra-individual length variation was identified and characterized in 15 species. The occurrence of heteroplasmy among species is phylogenetically consistent with a current classification of birds. Polymerase chain reaction amplifications, direct sequencing, and Southern analysis of mitochondrial DNA showed that the heteroplasmy is due to variable numbers of direct repeats in a tandem organization, located in the control region close to the tRNAPhe gene. The tandem repeats consist of short sequence motifs that vary in size from 4 to 32 base pairs between species. Sequence complexity of the repeat motifs was low, with almost exclusively Ts and Gs in the heavy-strand. Extensive variation in the copy number of the repeats was seen both intra-specifically and within individuals. This is the first report of mitochondrial heteroplasmy characterized at the sequence level in birds.

Animals↗

PubMedDate not supplied

Hypervariable 'minisatellite' regions in human DNA.

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

Alleles↗

PubMedDate not supplied

Copy number of cancer genes predict tumor grade and survival of pancreatic cancer patients.

Pancreatic cancer is on the increase. While means of early diagnosis are being sought, it continues to present late. Prognostication is based on patient and tumor characteristics, including expression or mutation of cancer-related genes. Few studies have examined the impact of the amplification of these genes on the outcome of pancreatic cancer. We have now used a non-radioisotopic slot-blot technique to relate gene copy numbers of p53, c-myc and K-ras to tumor grade and survival. Outcomes were corrected for patient characteristics, tumor location and TNM staging. The Kaplan-Meier test for likelihood of survival showed that increase in copy number of the two oncogenes and loss of p53 were associated with non-significant reduction in survival. When these variations in cancer gene copy numbers were, however, examined by logistic regression analysis in the context of patient and tumor characteristics, survival was negatively related to K-ras amplification (p = 0.0291). Tumor grade, but not survival was positively related to loss of p53 gene copy (p = 0.0131) as well as c-myc amplification (p = 0.0248). Thus using a simple non-radioisotopic technique for the detection of cancer gene copy number in association with patients and disease characteristics, we could predict survival on the one hand and tumor behavior on the other. Such information could be used to plan initial and follow-up therapy.

Adult↗

PubMed2001

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.

Amino Acid Sequence↗

PubMed1999

Amplification of a pseudogene cassette underlies euchromatic variation of 16p at the cytogenetic level.

Euchromatic imbalances at the cytogenetic level are usually associated with phenotypic consequences. Among the exceptions are euchromatic variants of chromosomes 8, 9, 15 and 16, which have each been reported in multiple unrelated families. In this paper, we present a new family and an unrelated individual who have euchromatic variants of 16p. Enhanced hybridisation to the extra material was found by using fluorescence in situ hybridisation with cosmids for both the 16p11.2-specific non-functional immunoglobin heavy chain segments and the pseudogenetic 16p11.2 creatine transporter region. Computerised measurement of the fluorescent signals was consistent with amplification of a pseudogene cassette comprising both these paralogous domains, which were originally transposed from 14q32.3 and Xq28, respectively. Amplification of pseudogenetic sequences is consistent with the normal phenotype in 36/46 carriers from the 18 families reported to date. Inconsistent phenotypic anomalies in the remaining 10 carriers probably reflect bias of ascertainment. These results are analogous to the amplification of the 15q11.2-specific pseudogene cassette in euchromatic variants of chromosome 15. They also suggest that the majority of established euchromatic variants are associated with variation in the copy number of sequences that have been dispersed between pericentromeric and telomeric loci over recent evolutionary time. We propose that constitutional cytogenetic amplification of this kind is part of a more widespread continuum of genomic flux affecting regions in which heterochromatin and euchromatin interpose. Euchromatic sequences that vary in a heterochromatic manner might usefully be termed "hemichromatic".

Adolescent↗

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

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus.

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3' end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, an estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1-4 million years ago.

Animals↗

PubMed1995

Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation.

A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped approximately using fragments from within EcoR1-I, and the precise location of the variable sites determined from the DNA sequence of this fragment. Oligonucleotide primers flanking the variable sites were synthesized, and used in PCR assays to detect variable fragments. The AluI variable fragment was found to result from the presence or absence of a single AluI site. In contrast, the variable bands seen with HaeIII and RsaI, resulted from variation in the copy number of two tandemly repeated sequences, one of which had not previously been recognized. In addition, HaeIII digests of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 also separated isolates into two groups. The variable HaeIII site was mapped towards the 5'-end of the gB gene and a PCR assay established. The distribution of the variable AluI site within the EcoR1-I fragment and the HaeIII site within the gB gene were estimated on a large number of clinical isolates using PCR on unpurified viral tissue culture medium. Both sites had a good distribution and together with additional variable sites should provide the basis for the rapid DNA fingerprinting of EHV-1 isolates.

Animals↗

PubMed1998

Copy number of pilus gene clusters in Haemophilus influenzae and variation in the hifE pilin gene.

Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (hifA to hifE) encoding pili similar to well-characterized Hif fimbriae of H. influenzae type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and the hifE(F3031) allele were special features of BPF-associated bacteria, we analyzed a collection of H. influenzae strains by PCR with hifA- and hifE-specific oligonucleotides, by Southern hybridization with a hifC gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some H. influenzae biogroup aegyptius strains. The hifE(F3031) allele was limited to H. influenzae biogroup aegyptius. Two strains contained one copy of hifE(F3031) and one copy of a variant hifE allele. We determined the nucleotide sequences of four hifE genes from H. influenzae biogroup aegyptius and H. influenzae capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable H. influenzae, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus of Neisseria pilus adhesin PilC. We concluded that two pilus clusters and hifE(F3031) were not specific for BPF-causing H. influenzae, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.

Adhesins, Bacterial↗

PubMed2002

Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer.

Cytogenetic analyses have revealed that many aneuploid breast cancers have cell-to-cell variations of chromosome copy numbers, suggesting that these neoplasms have instability of chromosome numbers. To directly test for possible chromosomal instability in this disease, we used fluorescent in situ hybridization to monitor copy numbers of multiple chromosomes in cultures of replicating breast cancer-derived cell lines and nonmalignant breast epithelial cells. While most (7 of 9) breast cancer cell lines tested are highly unstable with regard to chromosome copy numbers, others (2 of 9 cell lines) have a moderate level of instability that is higher than the "background" level of normal mammary epithelial cells and MCF-10A cells, but significantly less than that seen in the highly unstable breast cancer cell lines. To evaluate the potential role of a defective mitotic spindle checkpoint as a cause of this chromosomal instability, we used flow cytometry to monitor the response of cells to nocodazole-induced mitotic spindle damage. All cell lines with high levels of chromosomal instability have defective mitotic spindle checkpoints, whereas the cell lines with moderate levels of chromosomal instability (and the stable normal mammary cells and MCF10A cells) arrest in G(2) when challenged with nocodazole. Notably, the extent of mitotic spindle checkpoint deficiency and chromosome numerical instability in these cells is unrelated to the presence or absence of p53 mutations. Our results provide direct evidence for chromosomal instability in breast cancer and show that this instability occurs at variable levels among cells from different cancers, perhaps reflecting different functional classes of chromosomal instability. High levels of chromosomal instability are likely related to defective mitotic checkpoints but not to p53 mutations.

Breast Neoplasms↗

PubMed2002

Gene copy number regulates the production of the human chemokine CCL3-L1.

Genetic variation in the chemokine system is likely to affect responses to infection, and influence the course of autoimmune and inflammatory disease. We and others have shown that the human beta-chemokine CCL3-L1, unlike its related non-allelic isoform CCL3, has high affinity for the chemokine receptors D6, CCR3 and CCR5. Moreover, CCL3-L1, but not CCL3, is susceptible to cleavage by CD26, creating a truncated -2 form with enhanced affinity for CCR1 and CCR5. Strong interaction with CCR5 means that CCL3-L1, and particularly its -2 variant, are by far the most potent natural HIV entry inhibitors described to date. Here, using real-time PCR we have shown that CCL3-L1 and a novel CCL4 isoform (termed CCL4-L1) can vary from 1-6 copies per diploid genome (pdg) in Caucasians and are occasionally completely absent. The other isoforms (CCL3 and CCL4) remain at two copies per dpg. Importantly, in a model system of pro-inflammatory chemokine production (LPS-activated monocytes)higher gene copy number correlates with an increased ratio of CCL3-L1 versus CCL3 mRNA, and enhanced chemokine production. Supernatants from samples with high copy number are able to more potently chemoattract CCR5-expressing cells, an effect blocked with anti-CCL3/CCL3-L1 antibodies. As a result of these studies, we hypothesize that genetic variation in CCL3-L1 gene copy number may affect the susceptibility to, or the progression or severity of, diseases in which this chemokine plays a role.

Amino Acid Sequence↗

PubMed1997

Relational mapping of MYCN and DDXI in band 2p24 and analysis of amplicon arrays in double minute chromosomes and homogeneously staining regions by use of free chromatin FISH.

MYCN amplification has been observed in diverse neuronal tumors including neuroblastoma, retinoblastoma, and small cell carcinoma of the lung, and has been correlated with a poor prognosis in advanced-stage neuroblastomas. Recent studies have shown a co-amplification of DDXI, a DEAD box gene, and MYCN in retinoblastoma and neuroblastoma. DDXI has been mapped to within a megabase of the MYCN gene in band 2p24. In the present study, the relational map of DDXI and MYCN by fluorescence in situ hybridization (FISH) mapping to metaphase cells and extended free chromatin fibers indicated that DDXI is telomeric to MYCN. Dual-color FISH analysis of amplicons within arrays of extended chromatin fibers was performed to examine the physical relationship of MYCN and DDXI within double minute chromosomes (dmins) and homogeneously staining regions (hsrs). No regular reiterated amplicon repeat unit was present in the hsrs, but detailed analysis of the configurations of DDXI and MYCN within each array indicated that multiple rearrangements generated a complex hsr amplicon structure. Similarly, analysis of a cell line bearing dmins showed that a composite amplicon structure involving deletions and/or duplications of MYCN and DDXI is a feature of dmin formation. These data are consistent with a molecular mechanism involving many rearrangements during the evolution of gene amplification, resulting in complex amplicon structures with distinct changes in relative gene copy number and considerable variation in intragenic distances between coamplified genes.

Blotting, Southern↗

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

Computer image analysis of comparative genomic hybridization.

We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented.

Cell Line↗

PubMed2001

Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter.

A sequence of 1624 bp 5' to the apurinic/apyrimidinic (AP) endonuclease structural gene of Dictyostelium discoideum (APEA) has been inserted upstream of the luciferase reporter gene in pVTL2, an autonomously replicating nuclear plasmid in this organism. Cells transformed with this plasmid, designated pVTL-AL, displayed strong luciferase induction during treatment with the DNA-damaging agent bleomycin. For example, a luciferase activity of 45-fold above the constitutive level was observed for 20 hours of growth in axenic medium with 0.002 U/mL of bleomycin. The response was bleomycin concentration-dependent. Cell survival was greater than 90% for all treatments. The level of luciferase expression was highly dependent on the cell growth conditions, with the greatest induction observed for stationary phase axenically-grown cells. This effect may be related to a variation of plasmid copy number with growth conditions.

Animals↗

PubMed1991

A specific point mutation in the mitochondrial genome of Caucasians with MELAS.

The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) exhibits a specific heteroplasmic A----G transition in the tRNA(Leu) at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.

Acidosis, Lactic↗

PubMed1986

The evolution of the alpha- and beta-globin gene clusters in human populations.

DNA analysis of the alpha- and beta-globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous beta-thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of alpha-thalassaemia support the hypothesis that this condition, like the beta s-mutation, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.

Genetics, Population↗

PubMed1987

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins.

It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS+ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS+ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS+ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.

Amidohydrolases↗