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Results for “gene copy number”

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At least 19 recordsLinked to original sources

PubMed1990

Relationship of c-myc gene copy number and gene expression: cellular effects of elevated c-myc protein.

The relationship between gene copy number and expression and cellular consequences of elevated levels of c-myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c-myc gene. HC-8 and LC-5 recombinant cells carry approximately 800 and 50 copies of c-myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c-myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC-8 cells produced approximately tenfold more c-myc protein than heated LC-5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c-myc expression was observed following heat induction, even though the amounts of genomic c-myc were relatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c-myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c-myc protein in HC-8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c-myc protein are incompatible with cell survival.

Animals↗

PubMedDate not supplied

High-resolution analysis of gene copy number alterations in human prostate cancer using CGH on cDNA microarrays: impact of copy number on gene expression.

Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH) on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classic chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28) and loss (18) were found, and their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13q, and gains at 1q and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, and 74-76 Mbp from the p-telomere), which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, and 17q (losses), and at 3q, 5p, and 6p (gains). Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P <.0001) overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

Gene Dosage↗

PubMed1988

Transformation as a method of increasing gene copy number and gene expression in the basidiomycete fungus Coprinus cinereus.

Seven transformants having varying numbers of non-homologously integrated copies of the isocitrate lyase gene, acu-7, were analysed for enzyme activity. Maximum levels of activity, 3.8 times that of the wild type, were observed in a transformant with only two gene copies whereas eight gene copies in another transformant led to only 25% wild type activity. Acu+ transformants were not selected directly for expression of acu-7 but as cotransformants. Analysis of 14 transformants not expressing acu-7 showed that four contained transforming DNA sequences and significantly, two had evidence of non-homologously integrated tandem duplications of the entire acu-7 plasmid DNA. The site of integration of the gene was thus important in determining whether or not it was expressed and to what level it was expressed. A comparison of induced and uninduced levels of enzyme activity confirmed that the enzyme was still tightly regulated.

Agaricales↗

PubMed2006

Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri.

The metal hyperaccumulator Arabidopsis halleri exhibits naturally selected zinc (Zn) and cadmium (Cd) hypertolerance and accumulates extraordinarily high Zn concentrations in its leaves. With these extreme physiological traits, A. halleri phylogenetically belongs to the sister clade of Arabidopsis thaliana. Using a combination of genome-wide cross species microarray analysis and real-time reverse transcription-PCR, a set of candidate genes is identified for Zn hyperaccumulation, Zn and Cd hypertolerance, and the adjustment of micronutrient homeostasis in A. halleri. Eighteen putative metal homeostasis genes are newly identified to be more highly expressed in A. halleri than in A. thaliana, and 11 previously identified candidate genes are confirmed. The encoded proteins include HMA4, known to contribute to root-shoot transport of Zn in A. thaliana. Expression of either AtHMA4 or AhHMA4 confers cellular Zn and Cd tolerance to yeast (Saccharomyces cerevisiae). Among further newly implicated proteins are IRT3 and ZIP10, which have been proposed to contribute to cytoplasmic Zn influx, and FRD3 required for iron partitioning in A. thaliana. In A. halleri, the presence of more than a single genomic copy is a hallmark of several highly expressed candidate genes with possible roles in metal hyperaccumulation and metal hypertolerance. Both A. halleri and A. thaliana exert tight regulatory control over Zn homeostasis at the transcript level. Zn hyperaccumulation in A. halleri involves enhanced partitioning of Zn from roots into shoots. The transcriptional regulation of marker genes suggests that in the steady state, A. halleri roots, but not the shoots, act as physiologically Zn deficient under conditions of moderate Zn supply.

Adenosine Triphosphatases↗

PubMed2005

Determination of cytochrome P450 2D6 (CYP2D6) gene copy number by real-time quantitative PCR.

Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number). Using the 2(-Delta Delta Ct) calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies), and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

Journal Article↗

PubMed2000

Investigations on gene copy number, introns and chromosomal arrangement of genes encoding the fucoxanthin chlorophyll a/c-binding proteins of the centric diatom Cyclotella cryptica.

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.

Blotting, Southern↗

PubMed2003

Enhanced transgene expression in embryonal carcinoma stem cells: transcription through introns and exons increases gene copy numbers and forestalls silencing.

Efficient high-level expression of transfected genes in stem cells is difficult to achieve. Using P19 embryonal carcinoma stem cells, we previously showed that cotransfection with a fragment of the Pgk-1 gene increased the number of integrated copies of genes on cotransfected plasmids and resulted in enhanced expression of these genes. Active transcription through introns and exons of the Pgk-1-derived sequence was required for elevated expression of the cotransfected reporter genes. We show here that the enhanced expression appears to be due to two effects: (1) increased ligation of plasmid DNAs prior to integration into the host genome and (2) maintenance of an active domain in the region comprising the cluster of transgenes. We found that individual clones of stably transformed cells had highly variable numbers of transgenes integrated into their genomes but the ratio of genes comprising each transgene was proportional to the ratio of genes on different plasmids in the transfection mixture. The presence of the Pgk-1 gene fragment ensured that the expression of cotransfected genes in each clone was proportional to the numbers of integrated gene copies and selection for high-level expression of one cotransfected gene ensures high-level expression of the other cotransfected genes.

Animals↗

PubMed2000

Intron-exon structure and gene copy number of a gene encoding for a membrane-intrinsic light-harvesting polypeptide of the red alga Galdieria sulphuraria.

Genes for light-harvesting proteins (lhc genes) of higher plants are well examined. However, little is known about the corresponding genes of algae, although this knowledge might give valuable information about the evolution of photosynthetic antennae. In the case of rhodophytes only two cDNA sequences from a single organism, Porphyridium cruentum, have been published. Here we describe an additional sequence from another species, the thermo-acidophilic red alga Galdieria sulphuraria. For the first time also a genomic sequence for a red algal lhc gene is presented. From a cDNA library of G. sulphuraria we isolated a clone containing an open reading frame for a protein of 302 amino acids with a deduced molecular mass of 33.86kDa. It shares major structural features with eukaryotic light-harvesting polypeptides. A proposed cleavage site between transit peptide and mature protein gives rise to a transit peptide of 119 amino acids and a mature protein of 183 residues. Hydropathy analysis suggests that the mature protein consists of three transmembrane helices. Several amino acid residues supposed to bind chlorophyll a and chlorophyll b in higher plants are conserved. The protein shows up to 69% identity and 81% similarity to the Porphyridium polypeptides in the transmembrane helices 1 and 3. Using oligonucleotides annealing in the regions of the start and stop codons of the gene as primers, a DNA sequence was amplified from nuclear G. sulphuraria DNA by PCR. Compared with the cDNA clone, this sequence contains five additional intervening DNA strings of 50-74bp length. Four of them show typical features of spliceosomal introns with GT-AG borders, and the fifth differs by starting with GC. Three of the supposed introns are located in similar positions as introns of higher plant light-harvesting proteins. Southern blotting and hybridization experiments indicate that G. sulphuraria contains at least three copies of this gene.

Algal Proteins↗

PubMed1996

Expression of the immunoglobulin VH gene 51p1 is proportional to its germline gene copy number.

51p1 is an immunoglobulin VH gene that is frequently expressed in B cell chronic lymphocytic leukemia and early in B cell ontogeny. The 51p1 gene locus is highly polymorphic, consisting of 13 alleles that can be classified as being either 51p1-related or hy1263-related, based on distinctive sequence motifs in the second complementarity determining region. Two of the 51p1-related genes usually occur as a linked pair on the same haopltype, resulting from gene duplication. Consequently, a person can have a total of zero to four copies of 51p1-related genes. These genes are detectable in genomic DNA by sequence-specific RFLP analysis using oligonucleotide probes. Ig encoded by nonmutated 51p1-related genes can be detected by G6, a murine antiidiotypic mAb. We have now studied lymphocytes from 35 human tonsils to examine the relation between the number of 51p1-related germlime gene copies and the proportion of IgD-bearing tonsillar B cells that react with G6. All subjects who had zero copies of 51p1-related genes lacked any G6-reactive B cells, whereas those with four copies of 51p1-related genes had the highest proportions of G6-positive IgD B cells, up to 11.4%. Subjects with intermediate gene doses had intermediate proportions of G6-reactive B cells. Over the entire data set, the percentage of IgD-bearing B cells that reacted with G6 was proportional to the 51p1-related gene copy number (r = 0.92, p < 0.001), with each copy accounting for 2.4-4.0% of the IgD-bearing B cells. We conclude that 51p1-related genes are expressed by a relatively large percentage of IgD+ tonsillar B cells and this percentage is proportional to the germline copy number of 51p1-related genes.

Adult↗

PubMed2005

Detection of differential gene copy number using denaturing high performance liquid chromatography.

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.

Chromatography, High Pressure Liquid↗

PubMed2005

High-resolution gene copy number and expression profiling of human chromosome 22 in ovarian carcinomas.

Previous low-resolution studies of chromosome 22 in ovarian carcinoma have suggested its involvement in the development of the disease. We report a high-resolution analysis of DNA copy number and gene expression of 22q in 18 ovarian carcinomas using a 22q-specific genomic microarray. We identified aberrations in 67% of the studied tumors, which displayed 3 distinct gene copy number profiles. The majority of the cases (11 of 18) demonstrated heterozygous terminal deletions of various sizes, the smallest of which was 3.5 Mb. The second profile, detected in 3 tumors, revealed the coexistence of heterozygous deletions and different patterns of low-copy-number gain that involved the proximal half of 22q. The latter finding has not been reported previously in ovarian carcinoma. One case displayed a continuous deletion encompassing the entire 22q, consistent with monosomy 22. Furthermore, we compared the results with the available data on these tumors by using cDNA microarrays to define the degree of correlation between abnormalities at the DNA level and variation in mRNA expression. By a comparison with the expression data, we were able to identify 21 deleted genes showing low mRNA levels and 12 amplified genes displaying elevated gene expression, several of which play roles in cell cycle control and the induction of apoptosis. Our results indicated significant correlation between DNA copy number aberrations and variation in mRNA expression. We also identified several regions and candidate genes on 22q that should be studied further to determine their role in the development of ovarian cancer.

Adenocarcinoma, Clear Cell↗

PubMed1993

Improved quantitation of HER-2/neu gene copy number in breast tumor-derived DNA samples.

A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/neu gene copy numbers, permits visual quantitation of the tumors' gene copy numbers. The authors show that the HER-2/neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.

Autoradiography↗

PubMed2006

Gene copy number polymorphisms in an arbuscular mycorrhizal fungal population.

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus Glomus intraradices by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.

Base Sequence↗

PubMed2006

A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon.

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.

Chromosomes, Human, Pair 8↗

PubMed1998

Ribosomal RNA gene copy number and nucleolar-size polymorphisms within and among chicken lines selected for enhanced growth.

Ribosomal (r) DNA genotypes (rRNA gene copy number) and nucleolar phenotypes (nucleoli number and size) were studied in dam and sire commercial broiler pure lines from three primary breeder sources. Thirteen lines were studied to determine whether directionally selected broiler pure lines contain higher numbers of rRNA genes than a control line unselected for performance traits. Eight of the 13 lines exhibited rRNA gene copy averages between 261 and 331 copies, three lines had averages between 365 and 380, and two lines had average copy numbers equal to or greater than 450 rRNA genes. The overall source copy number average from one breeder company exhibited a value (402 rRNA genes) significantly different from the control value (300 rRNA genes). Nucleoli number and relative-size were examined in 9 of the 13 lines to establish ploidy and determine the population incidence of nucleolar size polymorphisms. All of the individuals examined for nucleolar phenotype expressed two nucleoli, indicating that gene copy number variation in those lines was generally unrelated to haploidy, aneuploidy, or polyploidy. A high frequency of individuals exhibited nucleolar size polymorphisms (line values of 57 to 87%). The results suggest that multiple nucleolus organizer region (NOR) types are segregating within and among broiler pure lines and that these NOR types contain variable numbers of rRNA genes that differ in nucleogenesis capacity.

Animals↗

PubMed1990

Differences in gene copy number carried by different MHC ancestral haplotypes. Quantitation after physical separation of haplotypes by pulsed field gel electrophoresis.

We have examined the hypothesis that MHC ancestral haplotypes have a specific content of genes regulating the extent of autoimmune reactions. Gene copy number was quantitated by objective densitometry after PFGE was used to separate heterozygous AHs of different lengths. Initially we analyzed examples of known gene copy number at the C4 and 21 hydroxylase loci and showed that the approach provides predictable results. We then studied heterozygotes containing one characterized and one uncharacterized AH with particular attention to the gene copy number at the C4, Cyp21, and DRB loci. Each AH studied has a characteristic gene copy number at each locus studied. The same may be true of TNF, but other possibilities must be considered. AHs are markers for extensive chromosomal segments including particular numbers of several functional genes. Since AHs mark susceptibility to autoimmune disease, differences in gene copy number may be implicated.

Blotting, Southern↗

PubMed2006

Clinicopathologic features and prognostic implications of epidermal growth factor receptor (EGFR) gene copy number and protein expression in non-small cell lung cancer.

Increased epidermal growth factor receptor (EGFR) gene copy numbers and mutations predict sensitivity to EGFR tyrosine kinase inhibitor in non-small cell lung cancer (NSCLC). However, the clinicopathologic features of EGFR gene copy status in NSCLC remain unclear. We retrospectively analyzed 262 cases of NSCLC, including 135 squamous cell carcinomas (SCC) and 112 adenocarcinomas (ADC), for which paraffin blocks of the resected primary lung mass were available. None had received EGFR-targeted therapy. EGFR gene copy number was evaluated using fluorescence in situ hybridization (FISH), and EGFR expression was determined immunohistochemically using a tissue microarray. A high EGFR gene copy (EGFR FISH-positive) was found in 30.2% of the cases (amplification in 11.1% and high polysomy in 19.1%). There was no significant difference in EGFR FISH status with respect to SCC and ADC histology. The EGFR FISH-positive rate was higher in non-smokers than in smokers in the multivariate analysis (p=0.012). EGFR expression was significantly associated with a high EGFR gene copy and SCC histology (p=0.000). In the univariate survival analysis, EGFR FISH-positivity predicted worse survival in SCC (p=0.059), especially stage I SCC (p=0.04). EGFR amplification was associated with a shorter survival in node-positive SCC (p=0.015). However, the EGFR gene copy number or protein expression had no influence on the prognosis of ADC. In conclusion, the EGFR FISH-positive rate in Korean patients with NSCLC was similar to rates in Western populations, unlike the higher frequencies of EGFR mutation in East Asians. A high EGFR gene copy number was significantly more common in non-smokers, as were EGFR mutations. A high EGFR gene copy number predicted worse survival in SCC; therefore, the prognostic implications of the EGFR gene and protein should be analyzed in the context of histology and staging in NSCLC.

Asian People↗

PubMed2006

A modified approach for I-FISH evaluation of ERBB2 (HER-2) gene copy numbers in breast carcinomas: comparison with HER-2/CEP17 ratio system.

PURPOSE: The evaluation of ERBB2 gene copy numbers and ERBB-2 protein expression in invasive duct carcinomas of the mammary gland (IDC) has been introduced as a part of a regular investigation protocol. Amplification of the ERBB2 gene detected by fluorescence in situ hybridization on interphasic nuclei (I-FISH) is used as a criterion for Herceptin administration. To improve characterization of cases with a borderline ERBB2 gene amplification, we applied a modified evaluation of the ERBB2 gene copy numbers. The results were compared with a commonly used HER-2/CEP17 ratio calculation. METHODS: We investigated 175 patients with primary IDCs in histological sections from paraffin embedded tissue with PathVysion HER-2 DNA Probe Kit (Vysis). Tumor cells of each case were sorted according to the number of ERBB2 signals into groups of tumor cells without amplification, with moderate amplification (< or =10 signals) and with strong amplification (>10 signals). If >10% of tumor cells had moderate or strong amplification of the ERBB2 gene, the case was reported as "moderately" or "strongly" amplified. RESULTS: The groups of patients classified by the proposed system as "without" and as with "strong" amplification had the HER-2/CEP17 ratio <1.91 (median 1.22, n = 33) and >2.3 (median 6.85, n = 115), respectively. Thus, the findings using the two systems of evaluation yielded similar results for these groups of patients. In cases, classified as with "moderate" amplification, the HER-2/CEP17 ratio varied from 1.3 to 4.77 (median 2.11, n = 27). Twelve of these 27 patients were according to the HER-2/CEP17 ratio system "not amplified" (1.3-1.93, median 1.72). Median percentage of the tumor cells with amplification of the ERBB2 gene was 14.5% in this subgroup. Of these 12 cases, 10 were ERBB-2 protein positive, and would be candidates for Herceptin therapy. CONCLUSION: The criteria for evaluation of the ERBB2 gene copy number proposed for this study separate gray zone cases with a borderline HER-2/CEP17 finding. The proposed system is easy to apply and characterizes a heterogeneity of the ERBB2 gene copy number in IDC tumor cell population more precisely than the currently used HER-2/CEP17 ratio system.

Breast Neoplasms↗