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Results for “DNA Copy Number Variations”

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PubMed1998

The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants.

Nuclear matrix attachment regions (MARs) are thought to influence gene expression by anchoring active chromatin to the proteinaceous nuclear matrix. In this study, two plant DNA fragments with strong MAR activity were selected and tested for their effects on expression of a linked reporter gene in transgenic tobacco. One MAR was isolated from the 5' flanking region of a pea vicilin gene previously reported to be expressed in a copy number-dependent manner in transgenic tobacco. A second MAR was isolated from the genome of Arabidopsis thaliana by preselection for autonomously replicating sequence (ARS) activity in yeast. Flanking copies of the A. thaliana MAR stimulated median reporter gene expression in transgenic plants by five to ten fold. Neither MAR significantly reduced the variation in transgene expression between individual transformants, or conferred copy number-dependence in gene expression.

Arabidopsis↗

PubMed1993

Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Base Sequence↗

PubMed1997

Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences.

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages.

Adaptation, Biological↗

PubMed2003

Diversity of group II introns in the genome of Sinorhizobium meliloti strain 1021: splicing and mobility of RmInt1.

The number and diversity of known group II introns in eubacteria are continually increasing with the addition of new data from sequencing projects, but the significance of these introns in the evolution of bacterial genomes is unknown. We analyzed the main features of the group II introns present in the genome of the soil microorganism Sinorhizobium meliloti (strain 1021), the nitrogen-fixing symbiont of alfalfa, the DNA sequence of which was recently determined. Strain 1021 harbors three different classes of group II introns: RmInt1, of bacterial class D; SMb2147/SMb21167, which cluster within bacterial class C; and SMa1875, the phylogenetic class of which is uncertain. The group II introns SMb2147/SMb21167 and SMa1875 are widely distributed in S. meliloti, but are present in lower copy numbers than RmInt1. Strain 1021 harbors three copies of RmInt1, which is pSym-specific. Although RmInt1 is spliced in strain 1021, mobility assays suggested that, in contrast to other S. meliloti strains, the genetic background of strain 1021 does not support intron homing events.

Bacterial Proteins↗

PubMed1994

Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago.

The primate Alu interspersed repeats can be subdivided into classes on the basis of shared nucleotides at a set of diagnostic positions. Each of the classes of Alu sequences is apparently the result of past retrotransposition of transcripts of highly conserved class-specific source genes that differed from each other at the diagnostic positions. The nucleotides at the majority of positions are identical among the source genes and therefore were identical among all of the Alu sequences at the time of their insertion. These CONSBI (conserved before insertion) positions are useful because the changes that have occurred after insertion are recognizable and the divergence resulting from nucleotide substitutions, insertions, and deletions is informative. The divergence of Alu sequences at the CONSBI positions is a measure of the time since a class was inserted. The greatest majority of Alu sequences are in one class (identified as class II), and it is particularly suitable for such examination, since nearly full-length sequences are now known for nearly a thousand members of this class. The average divergence of class II members indicates that the class has an average age of about 40 million years. The distribution in divergence of class II accurately fits a sum of two Poisson distributions. The implication is that class II Alu sequences were derived from two massive past events of insertion of many Alu sequences. In this model the younger subset of class II sequences (corresponding to about 300,000 copies in the genome) has an average divergence of 5% at CONSBI positions. The older set of class II sequences (corresponding to about 150,000 genomic copies) has a 9% average divergence. Based on the drift rate of primate DNA sequences, the events of insertion probably occurred 30-50 million years ago. The goodness of the fit to the Poisson distribution indicates that no significant number of members of class II have been inserted since 30 million years ago.

Animals↗

PubMed1994

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region.

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.

Journal Article↗

PubMed2002

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR.

OBJECTIVE: To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. METHODS: Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. RESULTS: Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon. CONCLUSIONS: It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Base Sequence↗

PubMed1990

Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster.

Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large.

Animals↗

PubMedDate not supplied

Giardia lamblia: identification and characterization of a variant-specific surface protein gene family.

Giardia lamblia trophozoites undergo antigenic variation by modulating the expression of variant-specific surface proteins (VSP), which are encoded by a number of small multigene families. We characterized the genomic copy of the VSP gene expressed by the cloned trophozoite line H7, derived from the isolate GS/M, in addition to a related, but nonexpressed, family member. The coding regions of the two genes encode closely related polypeptides (86% identity). However, differences in the coding region of these genes reside solely in an 873 bp segment. Only four differences were found between the 5' flanking sequences (465 bp). The proximal 205 base pairs downstream from the coding regions were identical, but thereafter the sequences diverged (37% identity over the next 391 bp). Mapping studies indicated that no other VSP gene was located within 4 kb pairs of the expressed H7 VSP gene, and transcripts from the nonexpressed gene were detected in neither GS/H7 nor heterogeneous trophozoites populations derived from this cloned line. Any mechanisms responsible for the differential expression of VSP genes must reconcile the near identity of DNA sequences that flank the coding regions of expressed and nonexpressed VSP genes.

Amino Acid Sequence↗

PubMed1989

Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations.

Several phenotypic variants of Halobacterium halobium arise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, the bop gene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes, gvpA and gvpC, are present on pNRC100, a multicopy plasmid of approximately 150 kilobase pairs. The gvpA and gvpC genes are on a single transcription unit, while the major bop mRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of the bop gene. Two elements, ISH 1 and ISH 2, account for most (80-90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number of gvpAC operon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗

PubMed2000

Copepod development rates in relation to genome size and 18S rDNA copy number.

It is known that body sizes and temperature-independent developmental durations within two genera of calanoid Copepoda (Crustacea) are positively related to nuclear DNA contents of their somatic nuclei. Evidently because of the constraint of similar cell numbers among the species, (nucleotypic) effects of nucleus size on cell size and on cell-level processes are expressed at the whole-organism level. Here, we show that developmental durations of eight species of five genera are also negatively related to their greatly differing numbers of 18S rRNA genes per unit DNA. We propose that levels of rDNA iteration among copepods have been controlled by natural selection to regulate ribosome concentrations, therefore protein production and development rates, independently of the large variations in genome sizes, which are in turn adapted to regulating cell and therefore body sizes.

Animals↗

PubMed1999

Wake up of transposable elements following Drosophila simulans worldwide colonization.

Transposable elements (TEs) make up around 10%-15% of the Drosophila melanogaster genome, but its sibling species Drosophila simulans carries only one third as many such repeat sequences. We do not, however, have an overall view of copy numbers of the various classes of TEs (long terminal repeat [LTR] retrotransposons, non-LTR retrotransposons, and transposons) in genomes of natural populations of both species. We analyzed 34 elements in individuals from various natural populations of these species. We show that D. melanogaster has higher average chromosomal insertion site numbers per genome than D. simulans for all TEs except five. The LTR retrotransposons gypsy, ZAM, and 1731 and the transposon bari-1 present similar low copy numbers in both species. The transposon hobo has a large number of insertion sites, with significantly more sites in D. simulans. High variation between populations in number of insertion sites of some elements of D. simulans suggests that these elements can invade the genome of the entire species starting from a local population. We propose that TEs in the D. simulans genome are being awakened and amplified as they had been a long time ago in D. melanogaster.

Animals↗

PubMed1988

The cDNA of cucumber mosaic virus-associated satellite RNA has in vivo biological properties.

Two isolates of cucumber mosaic virus (CMV)-associated satellite RNA, differing in their biological properties, have been reverse transcribed. One was able to induce the tomato necrotic syndrome whereas the other one attenuated fernleaf symptoms on tomato plants after co-inoculation with the helper virus. cDNAs representing partial or full-length copies have been cloned in the plasmid pAT 153 and sequenced. The two RNAs showed a very limited number of variations (2 to 5 substitutions depending on the clones and a one base deletion). Full-length cDNA copies possessed the same biological properties that characterized the parent satellite RNA. Efficiency of the cDNA depended upon its form in the inoculum (circular or linear plasmid or excised cDNA) and upon the form of the helper virus (viral RNAs or virions) with which it seemed to compete for installation and/or expression.

Base Sequence↗

PubMed2004

Quantification of multiple gene expression in individual cells.

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

Base Sequence↗

PubMed2004

Intratypic variability of a tandem repeat locus within the DNA polymerase gene of human herpes simplex virus type 2.

In this study, the irntratypic variability of a tandem repeat locus within the DNA polymerase (pol) gene of human herpes simplex virus type 2 (HSV2) was uncovered. The locus contained variable numbers of tandem dodecanucleotide (5'-GAC GAG GAC GGG-3') repetitive units. Our result showed that approximately 95% of analyzed HSV2 clinical isolates and the current GenBank HSV2 strains contained two copies of the repetitive units. From genital herpes specimens, three new HSV2 strains, which respectively contained 1, 3, and 4 copies of the repetitive units, were identified. This variable number of tandem repeat (VNTR) locus is absent in HSV1, and thus it also contributes to the intertypic variability of HSV1 and HSV2. The intratypic variability of the locus may be useful for HSV2 strain genotyping and this application is discussed.

DNA-Directed DNA Polymerase↗

PubMed1990

A partial map of the barley genome incorporating restriction fragment length polymorphism, polymerase chain reaction, isozyme, and morphological marker loci.

Nine low copy number genomic DNA clones, a ribosomal sequence, and seven cDNA clones were found to identify polymorphisms in cultivated barley (Hordeum vulgare L.). An F2 population consisting of 100 plants was produced from a cross between a high-yielding two-rowed feed barley cultivar and a genetic marker stock homozygous for nine recessive and one dominant morphological marker genes. Through the use of these 10 well-distributed marker genes, five previously mapped isozyme loci, and two storage-protein loci, the approximate recombinational location for each of 17 restriction fragment length polymorphism loci was estimated. One clone, pMSU21, identified variation that appeared to be the result of a small insertion-deletion event that differentiated two-rowed and six-rowed genotypes. This difference was characterized, and one allele was sequenced. Oligonucleotide primers that flanked the insertion-deletion event were synthesized, and DNA samples from the F2 population were subjected to polymerase chain reaction sequence amplification. The variation identified by this technique was determined to be allelic to the variation identified using pMSU21 in Southern blot analysis. Maps of previously undescribed informative clones are included.

Base Sequence↗

PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗

PubMed2003

[Transposition as a way of existence: phage Mu].

Transposition is a special mode of DNA recombination that produces a transfer of a DNA segment (transposable element, TE) from one chromosome region to a nonhomologous genomic loci. Bacterial TEs have chosen two principal strategies of their movement within and among genomes: conservative transposition (resulting in a simple insertion) and/or replicative transposition (accompanied by complete DNA replication and producing a cointegrate). Variations of transposition strategies concern the number and nature of cuts that sever the transposon from the flanking donor DNA: one-strand break (phage Mu) or double-strand breaks (Tn7, IS10 and members of IS3 family) and the possibility of accompanying TE replication, which leads to different end-products of transposition. Phage Mu combines properties of a temperate phage and a transposable element. Among transposons, it is studied in most detail. As a transposable element phage Mu is remarkable because its life-cycle involves two transposition modes: non-replicative resulting in lysogeny and replicative leading to multiple copying of phage DNA during the lytic growth. There are several levels of transcription regulation (or choice of developmental pathway). The genetic transposition reaction mediated by phage Mu A transposase A in the transpososome complex involves elaborate protein-nucleic acid and protein-protein interactions.

Animals↗