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Results for “Relative copy number variation”

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PubMed2005

Quantitative change in mitochondrial DNA content in various mouse tissues during aging.

In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.

Aging↗

PubMed2025

ELViS: an R package for estimating copy number levels of viral genomic segments at base-resolution.

MOTIVATION: Tumor viruses account for ∼10% of cancer diagnoses. Virally induced tumorigenesis is understood as direct signaling through oncogenes such as E6 and E7 genes in the case of human papillomavirus. Furthermore, pathogen characteristics such as viral oncogene dose may impact the disease course. To our knowledge, no tool has been proposed to assess the intra-viral copy number alterations that define the gene dose of viral oncogenes and associated suppressive pathways native to the pathogen's normal life cycle. RESULTS: We propose an R package, "ELViS," that analyzes viral copy number changes from DNA sequencing of whole viral genomes. The method adjusts for viral load with 2D transformation and segmentation to offer the relative viral gene doses. AVAILABILITY AND IMPLEMENTATION: The ELViS R package is available from https://bioconductor.org/packages/ELViS. This article used controlled access data from dbGaP (phs001713.v1.p1).

Software↗

PubMed1995

Selective DNA amplification regulates transcript levels in plant mitochondria.

Most plant mitochondrial genomes exist as subgenomic-size fragments apparently due to recombination between repetitive sequences. This leads to the possibility that independently replicating subgenomic domains could result in mitochondrial gene copy number variation. We show, through Southern-blot analysis of both restricted and intact mtDNA, that there are gene-specific copy number differences in the monocot Zea mays. Comparison of two different maize genotypes, B37(N) and B37(T), a cytoplasmic male-sterile strain, reveal fewer gene copy number differences for B37(T) than for B37(N). In contrast to maize, significant gene copy number differences are not detected in the dicot Brassica hirta. We also demonstrate that mitochondrial transcriptional rates in both species are apparently dependent on gene copy number since relative rates determined by run-on analysis are proportional to relative gene copy numbers. Thus a direct relationship exists between plant mitochondrial gene copy number and transcriptional rate.

Blotting, Southern↗

PubMed2000

Direct and indirect readout in mutant Met repressor-operator complexes.

BACKGROUND: The methionine repressor, MetJ, represses the transcription of genes involved in methionine biosynthesis by binding to arrays of two to five adjacent copies of an eight base-pair 'metbox' sequence. Naturally occurring operators differ from the consensus sequence to a greater extent as the number of metboxes increases. MetJ, while accommodating this sequence variation in natural operators, is very sensitive to particular base changes, even where bases are not directly contacted in the crystal structure of a complex formed between the repressor and consensus operator. RESULTS: Here we report the high-resolution structure of a MetJ mutant, Q44K, bound to the consensus operator sequence (Q44Kwt19) and two related sequences containing mutations at sites believed to be important for indirect readout at non-contacted bases. The overall structure of the Q44Kwt19 complex is very similar to the wild-type complex, but there are small variations in sugar-phosphate backbone conformation and direct contacts to the DNA bases. The mutant complexes show a mixture of direct and indirect readout of sequence variations, with differences in direct contacts and DNA conformation. CONCLUSIONS: Comparison of the wild-type and mutant repressor-operator complexes shows that the repressor makes sufficiently strong interactions with the sugar-phosphate backbone to accommodate some variation in operator sequence with minor changes in direct bases contacts. The reduction in repressor affinity for the two mutant repressor complexes can be partially attributed to a loss in direct contacts to the DNA. In one case, however, the replacement of a flexible TA base-step leads to an unfavourable DNA conformation that reduces the stability of the repressor-operator complex.

Amino Acid Sequence↗

PubMed1997

Repetitive sequence families in Alces alces americana.

High-resolution derivative melting was used to obtain detailed distributions of local (G + C) contents in a number of ruminant DNAs. Profiles over low (G + C) regions [20-36% (G + C)] are congruent for all ruminants. This region represents 45-50% of the nuclear DNA content and primarily contains intergenic and intron sequences. The high (G + C) region, where most coding sequences are found [38-68% (G + C)], is marked by satellite bands denoting the presence of transcriptionally inert, tandemly repetitive sequence families. These bands can be analyzed for the abundance, base composition, and sequence divergence of satellite families with relatively high precision. Band patterns are unique to each species; even closely related species can be readily distinguished by their base distribution profiles. Variations in nuclear DNA contents in ruminants, determined by flow cytometry, are primarily due to variations in abundances of these repetitive sequence families. Thus, A. alces (moose) is found to have 8.85 +/- 0.2 pg DNA/cell, 25% more than the average in ruminants, while the base distribution curve indicates the presence of an unusually abundant satellite of 52.6% (G + C). The size (1 kb) and sequence of this satellite corresponds to satellite-I of other cervids, and in consequence it is designated Alces-I. The sequence of a cloned repeat of Alces-I has a length of 968 bp, a (G + C) content of 52.6%, and contributes 35%, or almost 3 million copies to the nuclear DNA, exceeding by approximately 300% the average array size of this repeat family in related cervids. In situ hybridization indicates the repeat is distributed throughout centromeric regions of all 62 acrocentric autosomes. Alces-I has much greater-than-expected numbers of GG, GA, and AG and far fewer numbers of TA and CG duplets, characteristics of all tandem repeats. The sequence is judged to be orthologous with satellite-I sequences from Rangifer tarandus (caribou), Capreolus capreolus (roe deer), Muntiacus muntjac (Chinese muntjac) and Muntiacus reevesi (Indian muntjac), as well as Antilocapra americana (pronghorn), and the bovids Bos taurus and Ovis aries. A tentative tree for the five cervids is in excellent agreement with one proposed on the basis of morphological characteristics. Differences from a consensus sequence indicate transversions exceed transitions by almost twofold, suggesting that substitutions occur randomly, or nearly so.

Animals↗

PubMed1996

The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level.

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papl gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF.

Alanine↗

PubMed2001

Network analysis provides insights into evolution of 5S rDNA arrays in Triticum and Aegilops.

We have used network analysis to study gene sequences of the Triticum and Aegilops 5S rDNA arrays, as well as the spacers of the 5S-DNA-A1 and 5S-DNA-2 loci. Network analysis describes relationships between 5S rDNA sequences in a more realistic fashion than conventional tree building because it makes fewer assumptions about the direction of evolution, the extent of sexual isolation, and the pattern of ancestry and descent. The networks show that the 5S rDNA sequences of Triticum and Aegilops species are related in a reticulate manner around principal nodal sequences. The spacer networks have multiple principal nodes of considerable antiquity but the gene network has just one principal node, corresponding to the correct gene sequence. The networks enable orthologous groups of spacer sequences to be identified. When orthologs are compared it is seen that the patterns of intra- and interspecific diversity are similar for both genes and spacers. We propose that 5S rDNA arrays combine sequence conservation with a large store of mutant variations, the number of correct gene copies within an array being the result of neutral processes that act on gene and spacer regions together.

Algorithms↗

PubMed2003

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays.

BACKGROUND: Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear. RESULTS: Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 microm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. CONCLUSIONS: Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.

Arabidopsis↗

PubMed2005

High rate of horizontal transfer of transposable elements in Drosophila.

We have conducted molecular population genetics analyses to understand the relationships among the transposable elements (TEs) in Drosophila melanogaster, in combination with sequence comparisons of TEs from two related species, D. simulans and D. yakuba. We observed much lower than expected genetic differences among elements, clear evidence for departure from expectations for equilibrium copy numbers and little divergence between species. This suggests that a large proportion of TEs in D. melanogaster had a recent origin as a result of interspecies movement.

Animals↗

PubMed1994

The relationship between DNA structural variation and activities of P elements in P and Q strains of Drosophila melanogaster.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

Animals↗

PubMed2001

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

Algorithms↗

PubMed1997

Genetic variation in the dimorphic regions of RAP-1 genes and rap-1 loci of Babesia bigemina.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4+ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4+ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99%, nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3' ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.

Amino Acid Sequence↗

PubMed2006

Noise in protein expression scales with natural protein abundance.

Noise in gene expression is generated at multiple levels, such as transcription and translation, chromatin remodeling and pathway-specific regulation. Studies of individual promoters have suggested different dominating noise sources, raising the question of whether a general trend exists across a large number of genes and conditions. We examined the variation in the expression levels of 43 Saccharomyces cerevisiae proteins, in cells grown under 11 experimental conditions. For all classes of genes and under all conditions, the expression variance was approximately proportional to the mean; the same scaling was observed at steady state and during the transient responses to the perturbations. Theoretical analysis suggests that this scaling behavior reflects variability in mRNA copy number, resulting from random 'birth and death' of mRNA molecules or from promoter fluctuations. Deviation of coexpressed genes from this general trend, including high noise in stress-related genes and low noise in proteasomal genes, may indicate fluctuations in pathway-specific regulators or a differential activation pattern of the underlying gene promoters.

Flow Cytometry↗

PubMed2006

From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction.

Phylogenetic analyses of DNA sequences have prompted spectacular progress in assembling the Tree of Life. However, progress in constructing phylogenies among closely related species, at least for plants, has been less encouraging. We show that for plants, the rapid accumulation of DNA characters at higher taxonomic levels has not been matched by conventional sequence loci at the species level, leaving a lack of well-resolved gene trees that is hindering investigations of many fundamental questions in plant evolutionary biology. The most popular approach to address this problem has been to use low-copy nuclear genes as a source of DNA sequence data. However, this has had limited success because levels of variation among nuclear intron sequences across groups of closely related species are extremely variable and generally lower than conventionally used loci, and because no universally useful low-copy nuclear DNA sequence loci have been developed. This suggests that solutions will, for the most part, be lineage-specific, prompting a move away from 'universal' gene thinking for species-level phylogenetics. The benefits and limitations of alternative approaches to locate more variable nuclear loci are discussed and the potential of anonymous nongenic nuclear loci is highlighted. Given the virtually unlimited number of loci that can be generated using these new approaches, it is clear that effective screening will be critical for efficient selection of the most informative loci. Strategies for screening are outlined.

Cell Nucleus↗

PubMed1996

Expression of a bovine kappa-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette.

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine kappa-casein (kappa-CN) cDNA under the control of the goat beta-CN 5' promoter elements and 3' flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine kappa-CN RNA to the mammary gland and secretion of bovine kappa-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine kappa-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69%, and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine kappa-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine kappa-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did kappa-CN from bovine milk. A high degree of variation in the production of bovine kappa-CN within each of the transgenic lines was observed.

Amino Acid Sequence↗

PubMed1976

DNA reassociation kinetics in relation to genome size in four amphibian species.

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Triturus cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass. Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount. The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.

Amphibians↗

PubMed1988

Unequal partitioning of bovine mitochondrial genotypes among siblings.

Two polymorphic mitochondrial DNA genomes, differing by a single Hpa II restriction site, are present at significantly different levels in tissue of three sibling dairy cows. The relative ratio of the two heteroplasmic molecules varies 3-fold among these three animals and documents a rapid segregation of mitochondrial genotypes in mammals. DNA sequencing shows the difference is due to a single guanine at position 364 in bovine mitochondrial DNA. A model involving unequal partitioning of the two amplified mitochondrial DNA species during the early cell divisions of the embryo can explain the appearance of such variation in heteroplasmic sibling animals. The model provides a basis for understanding the rapid DNA sequence variation observed in vertebrate mitochondrial DNA despite its high copy number and strict maternal inheritance.

Animals↗

PubMed1997

Divergent transcriptional regulation among expanding human immunodeficiency virus type 1 subtypes.

The current AIDS pandemic represents the uneven spread of multiple genetically related subtypes (A to J) of human immunodeficiency virus type 1 (HIV-1). Notably, HIV-1 E in southeast Asia and HIV-1 C in sub-Saharan Africa are expanding faster and are likely of greater global significance than the HIV-1 B subtype prevalent in the United States and Europe. While many studies have focused on genetic variation among structural genes, we chose to conduct a comparative analysis of the long terminal repeats of HIV-1 E and HIV-1 C isolates and report subtype-specific differences in enhancer copy numbers and sequences, as well as divergent activation in response to the cellular transcriptional activators Rel-p65 and NFATc and viral Tat. This study is the first to identify functional distinctions in promoter architecture between HIV-1 subtypes and raises the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. Divergent transcriptional regulation may explain some of the epidemiologically observed differences in transmission and pathogenesis and underscores the need for further comparative analysis of HIV-1 regulation.

Base Sequence↗