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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↗

PubMed1991

[Detection of steroid 21-hydroxylase gene variation among normal Chinese and patients with congenital adrenal hyperplasia].

Genomic DNA from 40 unrelated healthy individuals and 16 families affected with 21-hydroxylase deficiency were digested with restriction enzyme Taq I followed by Southern blot and hybridization using a 21-hydroxylase (21-OH) gene cDNA probe. In healthy individuals the number of copies of 21-OHB gene was constant while that of 21-OHA gene was variable including deletion and duplication. In addition, we found an extra 5.6 kb band in one normal person and in one CAH family. Among the 16 affected families, 19% of 21-OHB gene were deleted. No homologous deletion was found. The frequency of deletion is lower than that reported in the literature. There was variation in the copy-number of 21-OHA gene among patients with CAH, yet no difference was found between the patients and normal persons. The information on prenatal diagnosis of CAH is briefly discussed.

Adrenal Hyperplasia, Congenital↗

PubMed2003

Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population.

Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.

Cell Culture Techniques↗

PubMed1998

Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number.

Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.

Bacillus subtilis↗

PubMed1974

Chromosomal variation and the origin of drug-resistant mutants in mammalian cell lines.

Drug-resistant mutants of somatic cell lines fall into two classes: one seems to correspond to classical gene mutation, the other is characterized, albeit transiently, by karyotypic instability, high reversion frequency, and low plating efficiency. An interpretation of the origin of these drug-resistant mutants is offered on the basis of chromosomal variation, which generates variation of the number of copies of each individual chromosome and, consequently, of gene dosage.

Alleles↗

PubMed1998

Multiple copies of the ALA-D gene are located at the Lv locus in Mus domesticus mice.

Incremental differences in delta-aminolevulinate dehydratase (ALA-D; the second enzyme of the heme biosynthetic pathway) activity among inbred mouse strains can be attributed to variation in the number of copies of the ALA-D gene. We have cloned and characterized the Lv locus from an inbred mouse strain (DBA/2J) that has three times the normal ALA-D activity levels. The entire 12-kb ALA-D gene plus 16 kb of flanking DNA are found in 28-kb tandemly repeating units. We used the derived nucleotide sequence surrounding the internal junction of the repeats to survey wild-caught mice and demonstrate that multiple copies of the ALA-D gene occur in 7 of 24 worldwide locations of Mus domesticus mice. Data are consistent with a model that high lead (Pb) in the environment may be providing a selective advantage to mice harboring multiple copies of the ALA-D gene, since the enzyme is potently inhibited by lead.

Animals↗

PubMed2000

Characterization of the topoisomerase I locus in human colorectal cancer.

DNA topoisomerase I (topo I) is the principle target for Camptothecin and its analogues. The topo I gene is located on chromosome 20q11.2-q13.1 and variation in topo I gene copy number has been shown to have impact on the in vitro sensitivity to topoisomerase I inhibitor chemotherapy. Fluorescence in situ hybridization (FISH) was used to detect and compare the TOPO I gene copy number between metaphase and interphase nuclei in a panel of 7 colorectal cancer cell lines. TOPO I gene copy number varied from 2 to 8 between cell lines, and signal in interphase nuclei demonstrated a linear relationship with that detected in metaphase nuclei. The structure of gene amplification included isochromosome formation, amplicon extension, and marker chromosome generation. Comparative genomic hybridization (CGH) was then used to further define the region of gain on chromosome 20. The region of gain contained the topo I gene and involved nearly all of 20q in most cases. This demonstrates a high degree of intrinsic variation in topo I gene copy number and the involvement of a 20q amplicon in colorectal cancer, which may have important implications for colorectal tumorigenesis and the use of chemotherapy.

Caco-2 Cells↗

PubMed2003

A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA.

New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.

Actins↗

PubMed1984

Variations in the length of human Y chromosome: a statistical study.

A statistical test is proposed to examine the relationship between the mean and variance of the relative length of Y chromosome (as measured by Y/A2 or Y/F indices) in various racial groups. It is shown that the variance of Y chromosome length within a population is quadratically related with its mean if the Y chromosome length variation is caused only by variations in the number of copies of repeated DNA sequences. No such relationship is observed in the available data on relative lengths of the Y chromosome in various racial groups. We thus conclude that the length polymorphism in human Y chromosome may not be solely due to variations in the amount of Y chromosome-specific repeated DNA.

Humans↗

PubMed2000

High CA repeat numbers in intron 13 of the endothelial nitric oxide synthase gene and increased risk of coronary artery disease.

Endothelial nitric oxide synthase (eNOS) plays a key role in vascular homeostasis. Because its product, nitric oxide, possesses vasodilatory and antiatherogenic properties, an altered eNOS function might promote atherosclerosis. We investigated the association between variations in CA repeat copy number [(CA), polymorphism] in intron 13 of the eNOS gene and the risk of coronary artery disease. (CA), polymorphism was investigated in 1000 consecutive patients with angiographically confirmed coronary artery disease and 1000 age- and gender-matched control subjects by a PCR-based fragment length calculation. Twenty-eight different alleles were identified containing 17-44 CA repeats. The presence of one allele containing > or = 38 repeats was associated with an excess risk of coronary artery disease (odds ratio 1.94, 95% confidence interval 1.31-2.86, P = 0.001). Carriers of alleles containing > or = 38 CA repeats were, in particular, overrepresented in the subgroup without common cardiovascular risk factors (odds ratio 3.39, 95% confidence interval 1.30-8.86, P = 0.009). Logistic regression analysis revealed that the (CA), polymorphism proved to be an independent risk factor (relative risk 2.17, 95% confidence interval 1.44-3.27, P = 0.0002). Our findings indicate that high numbers of CA repeats in intron 13 of the eNOS gene are associated with an excess risk of coronary artery disease.

Adenine↗

PubMed1990

Recurrent mutation of immunoglobulin and c-myc genes and differential expression of cell surface antigens occur in variant cell lines derived from a Burkitt lymphoma.

The phenotypic and molecular genetic characteristics of 4 variant sublines of the Burkitt lymphoma cell line Namalwa have been examined. The sublines are DNA-fingerprint-identical and derived from a monoclonal tumour, as shown by a rearrangement of the T-cell-receptor beta-chain gene common to the 4 sublines. There is non-co-ordinate expression of MHC class-I MHC class-II, surface immunoglobulin and a number of antigens recognized by CD MAbs on the different sublines. These different phenotypes of the cells are reminiscent of B cells arrested in varying states of cellular maturity. On Southern blots there are different patterns of restriction fragments hybridizing with Ig heavy- and light-chain gene probes among the sublines, indicating that multiple rearrangements or mutations of Ig genes have occurred in the cells. Different patterns of hybridizing fragments among the sublines were also found by using c-myc probes, implying the existence of different mutations of the c-myc locus. The c-myc mutation found in one of the sublines mapped to the 5' flanking sequence and in another 3' to the c-myc locus. Using the J17BS8 probe, which detects a restriction fragment length polymorphism in the 3' flanking region of the c-myc gene, a 4-fold variation in the gene copy number among the subline was found and one of the sublines was shown to be hemizygous for c-myc. Examination of DNA from early cultures of Namalwa cells showed that the alternations in Ig and c-myc structure had occurred on prolonged culture of the cells.

Antibodies, Monoclonal↗

PubMed2001

Copy-number fluctuation by unequal crossing-over in the chicken avidin gene family.

The chicken avidin gene (AVD) forms a closely clustered gene family together with several avidin-related genes (AVRs). In this study, we used fluorescence in situ hybridization on extended DNA fibers (fiber-FISH) to show that the number of the AVD and AVR genes differs between individuals. Furthermore, the gene copy-number showed wide somatic variation in white blood cells of the individuals. The molecular mechanism underlying the fluctuation is most probably unequal crossing-over and/or unequal sister chromatid exchange, as judged by the Gaussian distribution of the gene counts. By definition, an increase in gene number on one locus should be accompanied by a decrease on the other locus in unequal sequence exchange. The results suggest that copy-number lability may be more common among gene families than previously thought. The chicken avidin gene family also provides an excellent model for studying the mechanisms of recombination and gene conversion.

Animals↗

PubMed1989

Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer.

Variation in chromosomal DNA in Escherichia coli was studied with probes specific for the P-associated-pilus (pap) region. The presence of DNA homologous to pap was determined by dot blots. Variation in the number of copies of pap and in the organization of internal and flanking sequences was determined by Southern blot hybridization. The 229 strains studied were also classified by O:K:H serotyping and multilocus enzyme electrophoresis. There was considerable heterogeneity in the presence of pap and distribution of pap-homologous DNA in these E. coli strains from natural sources. In general, there was less variation in pap among strains of the same specific O:K:H serotype and enzyme electrophoretic type than among random isolates. There were, however, E. coli strains identified as members of the same clone by O:K:H serotyping and enzyme electrophoresis that were pap positive and pap negative or had different Southern blot patterns for the pap probes (pap type). There were also isolates of the same pap type that differed in two of three O:K:H serotype antigens and the majority of enzymes that determined their enzyme electrophoretic type. These latter two observations were interpreted as evidence for the horizontal (infectious) transfer of the pap-homologous sequences among clones of E. coli.

Bacterial Adhesion↗

PubMedDate not supplied

[Chromosomal anomalies in children with undifferentiated forms of metal retardation based on molecular cytogenetic research data].

The results of cytogenetic diagnosis of 1333 children with mental retardation and congenital malformations are presented. Chromosomal aberrations and chromosomal variants (1qh+, 9qh+, 16qh+) have been detected in 181 (13.9%) cases analyzed and in 158 (11.85%) cases, respectively. Molecular cytogenetic methods have been used for diagnosis or detalization of diagnosis in 42 cases from 181 (23.2%). Independent variation (or polymorphism) of copies number of "classical" and alpha-satellite DNA sequences, forming pericentromeric regions (1qh+, 9qh+, 16qh+) has been detected in patients with chromosomal variants.

Child↗

PubMed2004

Variation in RNA expression and genomic DNA content acquired during cell culture.

Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three. Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations. These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer. This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous.

Breast Neoplasms↗

PubMed2000

DNA copy number changes in Schistosoma-associated and non-Schistosoma-associated bladder cancer.

DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and non-schistosoma-associated (NSA) squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SA-SCC (P < 0. 01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways.

Aged↗