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PubMed1992

Heterogeneity in state and expression of viral DNA in polyoma virus-induced tumors of the mouse.

We have examined the state and expression of polyoma viral DNA in representative epithelial and mesenchymal tumors, using a combination of biochemical and in situ methods. Results showed wide variations among tumor types and also in different regions within individual tumors, with respect to copy number of viral DNA, presence or absence of deletions, and expression of early and late viral proteins. Epithelial tumors showed the greatest heterogeneity. High copy free viral DNA, frequently with deletions, was found in all such tumors. A portion of free viral DNA was recoverable as transcriptionally active minichromosomes. Three distinct subpopulations of cells were distinguished by in situ analyses. Type 1 cells showed high copy free viral DNA and expressed the major viral capsid protein VP1; these cells appeared to be at various stages of productive (lytic) viral infection. Some productively infected cells were able to undergo mitosis; in a portion of these cells, VP1 was found in close association with the mitotic spindle. Type 2 cells contained high copy free DNA but did not express VP1; by some unknown mechanism, these cells manifest a post-replication block to late gene expression and lytic infection. Type 3 cells contained only low copy, presumably integrated, viral DNA and expressed no VP1; they thus resemble cells transformed in vitro by the virus. Epithelial tumors contained variable mixtures of these subpopulations, while mesenchymal tumors were composed of Type 3 cells only. Differences in virus-cell interactions are discussed in terms of their possible implications in tumor development.

Animals↗

PubMed1983

Duplication and amplification of toxin genes in Vibrio cholerae.

Vibrio cholerae strains of the classical biotype all contain two widely separated copies of the cholera toxin operon ctxAB. In contrast, EI Tor strains containing multiple copies of ctx have their copies arranged on large tandem repeats which are either 7 or 9.7 kb in length. The variation in size among these large tandem duplications was due to a difference in the copy number of a smaller, 2.7 kb, tandemly repeated sequence (RS1) that is located at the novel joint of these duplications, as well as upstream and downstream of ctx. Southern blot hybridization analysis indicated that amplification of a DNA region carrying ctx and flanked by direct repeats of RS1 may be responsible for the hypertoxinogenic phenotype of EI Tor variants selected by intraintestinal growth in rabbits.

Animals↗

PubMed1998

Threshold expression of the tRNA(Lys) A8344G mutation in single muscle fibres.

We investigated the distribution in skeletal muscle of mitochondrial DNA (mtDNA) with the tRNA(Lys) A8344G mutation, which is associated with myoclonus epilepsy and ragged red fibres (MERRF) syndrome. Isolated muscle fibre segments (n = 144) from six individuals of two different families carrying the mutation were studied. Two of these individuals were affected by MERRF while four had no or minor clinical symptoms. In one individual with a low overall level of mutated mtDNA (mean 18%) the variation in the proportion of mutated mtDNA between individual muscle fibres ranged from 0 to 80%. This result demonstrates that segregation of the tRNA(Lys) A8344G mutation within a tissue may lead to very marked variation of the level of mutated mtDNA between individual cells. There was a very high apparent threshold level of mutated mtDNA (95.3-97.7%) for expression of histochemical cytochrome c oxidase (COX) deficiency in individual muscle fibres. The results indicated that this apparent threshold level varied slightly between patients. Ultrastructural examination revealed that an appreciable proportion of the mitochondria in COX-positive muscle fibres lacked COX activity. Variation in intercellular and interorganellar distribution of mutated mtDNA in addition to the absolute mtDNA copy number may explain differences in clinical phenotypes in patients with high levels of the tRNA(Lys) A8344G mutation.

Adolescent↗

PubMed2002

The evolution of the vertebrate beta-globin gene promoter.

Complexity analysis is capable of highlighting those gross evolutionary changes in gene promoter regions (loosely termed "promoter shuffling") that are undetectable by conventional DNA sequence alignment. Complexity analysis was therefore used here to identify the modular components (blocks) of the orthologous beta-globin gene promoter sequences of 22 vertebrate species, from zebrafish to humans. Considerable variation between the beta-globin gene promoters was apparent in terms of block presence/absence, copy number, and relative location. Some sequence blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Block similarities were also evident between the promoters of the paralogous human beta-like globin genes. It may be inferred that a wide variety of different mutational mechanisms have operated upon the beta-globin gene promoter over evolutionary time. Because these include gross changes such as deletion, duplication, amplification, elongation, contraction, and fusion, as well as the steady accumulation of single base-pair substitutions, it is clear that some redefinition of the term "promoter shuffling" is required. This notwithstanding, and as previously described for the vertebrate growth hormone gene promoter, the modular structure of the beta-globin promoter region and those of its paralogous counterparts have continually been rearranged into new combinations through the alteration, or shuffling, of preexisting blocks. Some of these changes may have had no influence on promoter function, but others could have altered either the level of gene expression or the responsiveness of the promoter to external stimuli. The comparative study of vertebrate beta-globin gene promoter regions described here confirms the generality of the phenomenon of sequence block shuffling and thus supports the view that it could have played an important role in the evolution of differential gene expression.

Amino Acid Sequence↗

PubMed1992

Use of a cis-acting mutation to study the role of FLP-mediated recombination in the maintenance of native yeast 2 micrometer plasmids.

The 2 micrometer plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site-specific recombination system that is thought to be involved in plasmid copy-number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2 micrometer variant that has a cis-acting mutation in the FLP-mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP-mediated amplification compensates for this and therefore has an important role in maintenance.

DNA Nucleotidyltransferases↗

PubMed2002

The ner gene of Photorhabdus: effects on primary-form-specific phenotypes and outer membrane protein composition.

The nematode-bacterium complex of Heterorhabditis-Photorhabdus is pathogenic to insect larvae. The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes. The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries. Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as ner, a regulatory gene in gram-negative bacteria and their phages. Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form. These and other pieces of evidence indicate that the endogenous ner gene is not responsible for the secondary phenotype, but that ner, when overexpressed, can repress expression of primary phenotypes at stationary phase. Inactivation of the endogenous ner gene in the primary form affected the outer membrane protein profile. A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the ner primary strain.

Amino Acid Sequence↗

PubMed2006

Medical applications of array CGH and the transformation of clinical cytogenetics.

Microarray-based comparative genomic hybridization (array CGH) merges molecular diagnostics with traditional chromosome analysis and is transforming the field of cytogenetics. Prospective studies of individuals with developmental delay and dysmorphic features have demonstrated that array CGH has the ability to detect any genomic imbalance including deletions, duplications, aneuploidies and amplifications. Detection rates for chromosome abnormalities with array CGH range from 5-17% in individuals with normal results from prior routine cytogenetic testing. In addition, copy number variants (CNVs) were identified in all studies. These CNVs may include large-scale variation and can confound the diagnostic interpretations. Although cytogeneticists will require additional training and laboratories must become appropriately equipped, array CGH holds the promise of being the initial diagnostic tool in the identification of visible and submicroscopic chromosome abnormalities in mental retardation and other developmental disabilities.

Chromosome Aberrations↗

PubMed1992

Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells.

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.

Animals↗

PubMed2003

Interference of contaminating DNA in the quantification of a toluene-induced tod gene in Pseudomonas putida.

To determine the extent of interference of co-extracted DNA contamination in the quantification of the tod gene transcript, two different concentrations of RNA (high, 500 ng/microl; low, 250 ng/microl) from a toluene-induced culture of Pseudomonas putida were treated with different amounts of DNase (2, 4, 6 and 8 U) and incubated for 30 and 60 min. The highly sensitive and reproducible TaqMan system was used to quantify the transcript of the tod gene, the tod gene in contaminating DNA and the 16S rRNA gene in DNase-treated RNA samples. For the high RNA concentration, the shorter incubation time (30 min) lowered the level of contaminating DNA as evidenced by the presence of 2.5 x 10(6) copies of the tod gene before treatment to 1.4 x 10(5) copies/microl (8 U), whereas, irrespective of the DNase units used, the longer incubation time (60 min) considerably lowered the level of DNA contamination (2.5 x 10(6) to 6.5 x 10(2) copies of the tod gene/microl). However, for the low RNA concentration, DNase treatment was found to be equally effective in lowering the level of contaminating DNA (10(6) to 10(2) copies of the tod gene/mu), irrespective of the incubation time and the amount of DNase used. Although the results of gel electrophoresis of conventional PCR amplification of the low RNA concentration revealed the absence of the target gene in contaminating DNA, the results of the TaqMan PCR indicated that a very low amount of contaminating DNA (less than 10(3) copies of the tod gene/mul) was still present in RNA samples, even after the DNase treatment. The number of copies of the tod gene transcript in RNA samples did not show any marked variation because of the DNase treatment. However, the proportion of contaminating DNA in RNA samples considerably decreased due to the treatment (0.01 to 0.000001). Furthermore, these results suggested that the extent of the removal of contaminating DNA from RNA samples depends on the concentration of RNA, the amount of DNase used and the incubation time. It is also suggested that the copies of the catabolic genes in contaminating DNA have to be quantified along with the target genes in RNA samples to have a more accurate quantification of the target genes for better understanding of their roles in many microbial processes.

Journal Article↗

PubMed2006

SMN mRNA and protein levels in peripheral blood: biomarkers for SMA clinical trials.

BACKGROUND: Clinical trials of drugs that increase SMN protein levels in vitro are currently under way in patients with spinal muscular atrophy. OBJECTIVE: To develop and validate measures of SMN mRNA and protein in peripheral blood and to establish baseline SMN levels in a cohort of controls, carriers, and patients of known genotype, which could be used to follow response to treatment. METHODS: SMN1 and SMN2 gene copy numbers were determined in blood samples collected from 86 subjects. Quantitative reverse transcription PCR was used to measure blood levels of SMN mRNA with and without exon 7. A cell immunoassay was used to measure blood levels of SMN protein. RESULTS: Blood levels of SMN mRNA and protein were measured with high reliability. There was little variation in SMN levels in individual subjects over a 5-week period. Levels of exon 7-containing SMN mRNA and SMN protein correlated with SMN1 and SMN2 gene copy number. With the exception of type I SMA, there was no correlation between SMN levels and disease severity. CONCLUSION: SMN mRNA and protein levels can be reliably measured in the peripheral blood and used during clinical trials in spinal muscular atrophy, but these levels do not necessarily predict disease severity.

Biomarkers↗

PubMed2005

Variation of the Mycobacterium tuberculosis PE_PGRS 33 gene among clinical isolates.

PE_PGRS 33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS 33 genes of H37Rv and CDC 1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS 33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS 6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS 33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS 33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS 33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS 33 gene among clinical isolates.

Antigens, Bacterial↗

PubMed2003

Heterogeneous copy numbers of API2-MALT1 chimeric transcripts in mucosa-associated lymphoid tissue lymphoma.

T(11;18)(q21;q21) results in a chimeric transcript between API2 at 11q21 and MALT1 at 18q21 and is a characteristic chromosomal aberration of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). API2-MALT1 chimeric transcripts are present in approximately one-third of all cases of MALT lymphoma. MALT lymphoma is also known to have variations in histological features and tumor cell proportions. Real-time polymerase chain reaction (PCR) was used to examine number of API2-MALT1 copies in clinical samples for further investigation of the pathophysiology of MALT lymphoma. A total of 13 samples of MALT lymphoma contained API2-MALT1 transcripts from 1.7 x 10(-2) to 1.0 copies/beta-actin copy. These findings were compared to the proportions of tumor cells in genomic VDJ PCR products determined by Southern blotting. Tumor cell ratios varied widely among the patients' samples, and no significant correlation was found between transcript copy number and tumor cell ratio. These results suggest that copy numbers of API2-MALT1 do not reflect tumor cell proportions, and that the number of copies of API2-MALT1 in a tumor cell is different for each clinical sample.

Adult↗

PubMed1988

Independent variations of myc amplification, inducibility, maturation, and proliferation states in HL60.

We have investigated the possible relationship between c-myc gene activity and other variable traits in HL60. In a panel of variant lines, a good correlation was observed between myc gene copy number and the level of myc mRNA. There was no correlation between myc amplification or expression and the resistance of the lines to induction of terminal neutrophilic or monocytic differentiation. Therefore, myc mRNA level does not appear to determine the ability of the variant HL60 lines to respond to inducers of differentiation. Flow cytometric analyses of the expression of a differentiation antigen (AGF 4.36) revealed stable negative and positive subpopulations in growing HL60. myc amplification, expression, and inducibility were identical in these subpopulations, suggesting that variation of these traits in HL60 sublines and variants is not due to maturation state differences. myc gene copy number was also identical in transferrin receptor positive (proliferating) and negative (resting) populations. These data contradict the notion that myc amplification has been important in determining the in vitro biological properties of HL60.

Cell Division↗

PubMed1992

The population biology and evolutionary significance of Ty elements in Saccharomyces cerevisiae.

The basic structure and properties of Ty elements are considered with special reference to their role as agents of evolutionary change. Ty elements may generate genetic variation for fitness by their action as mutagens, as well as by providing regions of portable homology for recombination. The mutational spectra generated by Ty1 transposition events may, due to their target specificity and gene regulatory capabilities, possess a higher frequency of adaptively favorable mutations than spectra resulting from other types of mutational processes. Laboratory strains contain between 25-35 elements, and in both these and industrial strains the insertions appear quite stable. In contrast, a wide variation in Ty number is seen in wild isolates, with a lower average number/genome. Factors which may determine Ty copy number in populations include transposition rates (dependent on Ty copy number and mating type), and stabilization of Ty elements in the genome as well as selection for and against Ty insertions in the genome. Although the average effect of Ty transpositions are deleterious, populations initiated with a single clone containing a single Ty element steadily accumulated Ty elements over 1,000 generations. Direct evidence that Ty transposition events can be selectively favored is provided by experiments in which populations containing large amounts of variability for Ty1 copy number were maintained for approximately 100 generations in a homogeneous environment. At their termination, the frequency of clones containing 0 Ty elements had decreased to approximately 0.0, and the populations had became dominated by a small number of clones containing > 0 Ty elements. No such reduction in variability was observed in populations maintained in a structured environment, though changes in Ty number were observed. The implications of genetic (mating type and ploidy) changes and environmental fluctuations for the long-term persistence of Ty elements within the S. cerevisiae species group are discussed.

Biological Evolution↗

PubMed1997

Wide variation in DNA content among isolates of Trypanosoma brucei ssp.

The DNA contents of 18 Trypanosoma brucei ssp. stocks were compared using flow cytometry, karyotype analysis and quantitation of repetitive DNA by Southern blotting and hybridisation. The DNA contents of Type 1 T. b. gambiense stocks were lower than those of non-gambiense stocks, but both groups showed a wide range of variation in DNA content. Amongst T. b. gambiense stocks. Mabia at the lower end of the range had 14% less DNA than Dal 972 at the top of the range. Similarly, amongst non-gambiense stocks. 117R at the lower end of the range had 14% less DNA than LM55 at the top of the range. The T. b. gambiense stock Mabia had 29% less DNA than non-gambiense stock LM55. The DNA content of Type II T. b. gambiense stocks had minichromosomes albeit fewer than non-gambiense stocks. This result was verified by hybridisation with probes for satellite DNA and a telomere-specific repeat. Hybridisation with the probe for the beta-tubulin genes also revealed an apparent reduction of gene copy number T. b. gambiense relative to non-gambiense stocks. In conclusion, there is a wide range of variation in genome size in T. brucei ssp., with T. b. gambiense stocks at the lower end of the range. The reduction in genome size correlates with loss of repeated genes and non-coding sequences in T. b. gambiense stocks, and is not continued to chromosomes of a particular size.

Animals↗

PubMed1998

Intra- and inter-specific variation in nuclear ribosomal internal transcribed spacer 1 of the Schistosoma japonicum species complex.

The first internal transcribed spacer (ITS1) of the nuclear ribosomal DNA repeat was sequenced for members of the Schistosoma japonicum species complex (S. malayensis, S. mekongi and 2 geographical isolates of S. japonicum). The ITS1 is composed of 3 distinct regions: the 5' end (23 nucleotides); a tract of approximately 90-140 nucleotides, which occurs up to 7 times in tandem, the number varying even within an individual in all species investigated in this study; the 3' region (378 nucleotides), which lacks repeats. There is size and sequence variation among copies of the ITS1 repeat within a single individual. The relative abundances of size variants of ITS1 in S. japonicum have been ascertained by hybridizing genomic digests with an ITS1 probe. Multiple repeats and intra-individual variation in numbers and abundance of these is a feature of the Asian schistosomes, but not generally of African schistosomes. Possible reasons for this difference in ITS1 between African and Asian schistosomes are discussed. The ITS1 repeat sequences described for African schistosomes are different to, and cannot be aligned with, those from the Asian species described here, whereas the remainder of the ITS1 can be aligned quite easily.

Animals↗

PubMed2007

Prediction of chromosomal aneuploidy from gene expression data.

Chromosomal aneuploidy is commonly observed in neoplastic diseases and is an important prognostic marker. Here we examine how gene expression profiles reflect aneuploidy and whether these profiles can be used to detect changes in chromosome copy number. We developed two methods for detecting such changes in the gene expression profile of a single sample. The first method, fold-change analysis, relies on the availability of gene expression data from a large cohort of patients with the same disease. The expression profile of the sample is compared with that of the dataset. The second method, chromosomal relative expression analysis, is more general and requires the expression data from the tested sample only. We found that the relative expression values are stable among different chromosomes and exhibit little variation between different normal tissues. We exploited this novel finding to establish the set of reference values needed to detect changes in the copy number of chromosomes in a single sample on the basis of gene expression levels. We measured the accuracy of the performance of each method by applying them to two independent leukemia datasets. The second method was also applied to two solid tumor datasets. We conclude that chromosomal aneuploidy can be detected and predicted by analysis of gene expression profiles. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Aneuploidy↗

PubMed1983

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility.

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.

Animals↗