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Results for “DNA Copy Number Variations”

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PubMed1998

Unusual and strongly structured sequence variation in a complex satellite DNA family from the nematode Meloidogyne chitwoodi.

An AluI satellite DNA family has been isolated in the genome of the root-knot nematode Meloidogyne chitwoodi. This repeated sequence was shown to be present at approximately 11,400 copies per haploid genome, and represents about 3.5% of the total genomic DNA. Nineteen monomers were cloned and sequenced. Their length ranged from 142 to 180 bp, and their A + T content was high (from 65.7 to 79.1%), with frequent runs of As and Ts. An unexpected heterogeneity in primary structure was observed between monomers, and multiple alignment analysis showed that the 19 repeats could be unambiguously clustered in six subfamilies. A consensus sequence has been deduced for each subfamily, within which the number of positions conserved is very high, ranging from 86.7% to 98.6%. Even though blocks of conserved regions could be observed, multiple alignment of the six consensus sequences did not enable the establishment of a general unambiguous consensus sequence. Screening of the six consensus sequences for evidence of internal repeated subunits revealed a 6-bp motif (AAATTT), present in both direct and inverted orientation. This motif was found up to nine times in the consensus sequences, also with the occurrence of degenerated subrepeats. Along with the meiotic parthenogenetic mode of reproduction of this nematode, such structural features may argue for the evolution of this satellite DNA family either (1) from a common ancestral sequence by amplification followed by mechanisms of sequence divergence, or (2) through independent mutations of the ancestral sequence in isolated amphimictic nematode populations and subsequent hybridization events. Overall, our results suggest the ancient origin of this satellite DNA family, and may reflect for M. chitwoodi a phylogenetic position close to the ancestral amphimictic forms of root-knot nematodes.

Animals↗

PubMed1989

The molecular epidemiology of three biotypes of Corynebacterium diphtheriae in the Seattle outbreak, 1972-1982.

Between 1972 and 1982 Seattle experienced a diphtheria outbreak involving 1,100 cases, primarily adults with cutaneous lesions. Biotyping revealed three consecutive overlapping outbreaks including 433 toxinogenic intermedius cases. Isolates from each quarter year were examined for DNA restriction fragment patterns and hybridization patterns with three DNA probes. All intermedius outbreak isolates appeared identical in all analyses and most stock cultures had the outbreak DNA fragment pattern, but DNA probes detected six patterns within 11 intermedius stock cultures. The mitis outbreak was heterogeneous, involving at least five restriction fragment patterns. In contrast, DNA restriction fragment analyses indicated that 22 of 25 gravis outbreak isolates belonged to a single strain, whereas there were seven strains within eight gravis stock cultures. DNA probe analyses of gravis outbreak isolates detected seven different patterns, five involving copy numbers of the toxB fragment and attB sites that presumably reflected lysogenic events that occurred during the outbreak.

Bacterial Typing Techniques↗

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↗

PubMed2001

[Genetic instability in cancer tissues analyzed by RAPD PCR].

In five kinds of tumors, total 128 specimens were analyzed by RAPD (random amplified polymorphic DNA) PCR with nine 10-base arbitrary primers for detecting instabilities of DNA and chromosome and screening new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Bands representing instabilities have been recovered and purified from agarose and cloned into pCAPs vector, and further labeled by DIG as probes for analysis of Southern blot, Northern blot and Sequencing. Results revealed that sample 5 and 3 of the gastric cancers showed the highest genomic changes and the average detectability in five sorts of cancers was up to at least 40% (42.2%-49.4%), and that there were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% to 68%. Despite the highest detectability of genetic instability (68%) in tumor tissues, primer 2 could produce stable profiles of DNA bands in normal tissue genome with good reproducibility. On the contrary, primer 8 was of the lowest one (27%). Band B of single copy found to be allelic losses in gastric and colon cancers according to RFLP analysis was of a novel sequence and registered by Gen-Bank (Accession Number AF151005). Therefore the genetic instabilities often concentrated on some special locuses of chromosome e.g. repetitive sequences etc. and coupled to carcinogenesis. 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↗

PubMed1986

The evolution of DNA sequences in Escherichia coli.

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.

Base Sequence↗

PubMed2005

Chompy: an infestation of MITE-like repetitive elements in the crocodilian genome.

Interspersed repeats are a major component of most eukaryotic genomes and have an impact on genome size and stability, but the repetitive element landscape of crocodilian genomes has not yet been fully investigated. In this report, we provide the first detailed characterization of an interspersed repeat element in any crocodilian genome. Chompy is a putative miniature inverted-repeat transposable element (MITE) family initially recovered from the genome of Alligator mississippiensis (American alligator) but also present in the genomes of Crocodylus moreletii (Morelet's crocodile) and Gavialis gangeticus (Indian gharial). The element has all of the hallmarks of MITEs including terminal inverted repeats, possible target site duplications, and a tendency to form secondary structures. We estimate the copy number in the alligator genome to be approximately 46,000 copies. As a result of their size and unique properties, Chompy elements may provide a useful source of genomic variation for crocodilian comparative genomics.

Alligators and Crocodiles↗

PubMed2000

Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR.

We took advantage of a recently developed system allowing performance of real-time quantitation of polymerase chain reaction to develop a quantitative method of measurement of PML-RARalpha transcripts which are hallmarks of acute promyelocytic leukemia (APL) with t(15;17) translocation. Indeed, although quantitation of minimal residual disease has proved to be useful in predicting clinical outcome in other leukemias such as chronic myeloid leukemia or acute lymphoblastic leukemia, no quantitative data have been provided in the case of APL. We present here a method for quantitation of the most frequent subtypes of t(15;17) transcripts (namely bcr1 and bcr3). One specific forward primer is used for each subtype in order to keep amplicon length under 200 bp. The expression of PML-RARalpha transcripts is normalized using the housekeeping porphobilinogen deaminase (PBGD) gene. This technique allows detection of 10 copies of PML-RARalpha or PBGD plasmids, and quantitation was efficient up to 100 copies. One t(15;17)-positive NB4 cell could be detected among 106 HL60 cells, although quantitation was efficient up to one cell among 105. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay variation coefficients were not higher than 15%. The efficiency of the method was finally tested in patient samples, showing a decrease of the PML-RARalpha copy number during therapy, and an increase at the time of relapse.

Chromosomes, Human, Pair 15↗

PubMed1995

Structure, inheritance, and transcriptional effects of Pce1, an insertional element within Phanerochaete chrysosporium lignin peroxidase gene lipI.

A 1747-bp insertion within a lignin peroxidase allele of Phanerochaete chrysosporium BKM-F-1767 is described. Pce1, the element, lies immediately adjacent to the fourth intron of lip12. Southern blots reveal the presence of Pce1-homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (one to five copies), and conserved transposase motifs are lacking. The mutation transcriptionally inactivates lip12 and is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element that may play a significant role in generating ligninolytic variation in certain P. chrysosporium strains.

Alleles↗

PubMedDate not supplied

Allele-specific variation in the gene copy number of human cytosine 5-methyltransferase.

Previously, we have identified two alternate allelic forms of cytosine 5-methyltransferase, 5-MT I and 5-MT II, specified by polymorphic fragments of 1.5 and 1.1 kb, respectively. In the presence study, a 0.8-kb genomic probe was prepared which was confirmed to be included within the polymorphic fragments. The 0. 8-kb probe hybridised with greater intensity to the 1.1-kb fragment than the 1.5-kb fragment. Densitometric analysis indicated that there is 1 copy of 5-MT associated with 5-MT I, whereas there may be 1-4 copies of the gene associated with the 5-MT II allele. Segregation studies demonstrated that the multiple copies of 5-MT II are inherited in a Mendelian fashion. These results allow novel approaches to investigating the underlying mechanisms of cytosine methylation and gene duplication.

Alleles↗

PubMed2006

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization.

PURPOSE: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. METHODS: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification. RESULTS: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%. CONCLUSION: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.

Chromosome Aberrations↗

PubMed2005

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE.

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

DNA Primers↗

PubMed2003

Diversity of aspartate carbamoyltransferase genes of Trypanosoma cruzi.

The pyrimidine-biosynthetic (pyr) gene cluster, a tandem array of pyr1-pyr3-pyr6/5-pyr2(ACT)-pyr4 from the 5' terminus, encodes all the six enzymes of de novo pyrimidine biosynthesis and occurs as a polycistronic transcription unit in Trypanosoma cruzi. The gene encoding aspartate carbamoyltransferase (ACT), the second enzyme of the pathway, was characterised using a laboratory-reared Tulahuen strain and Tulahuen-derived clones of T. cruzi. Three loci with different restriction maps that contain ACT1, ACT2, and ACT3 were identified. ACT1 and ACT2 are involved in the pyr gene cluster on two different chromosomal DNA molecules of 1,000 and 800 kb, respectively, whereas ACT3 is linked with pyr4 alone. There are 29 nucleotide substitutions out of 981 positions in these three ACTs, yielding 13 amino acid replacements, and a deletion of triplet nucleotides in ACT1 entails a lack of single amino acid residue. Transcription of the three ACTs takes place in the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. Pulsed field gel electrophoresis and Southern blot analyses demonstrated that the cloned T. cruzi stocks, Y-02, CAN III/1, Sylvio-X10/4, and possibly Esmeraldo/3, also possess two complete sets of the pyr gene cluster including ACT, accompanied by additional incomplete clusters. These results suggest that the marked intra-species diversity in the copy number and chromosomal localisations of ACT and other pyr genes may have resulted from partial duplications and subsequent translocations of the polycistronic pyr gene cluster.

Amino Acid Sequence↗

PubMed1987

Mutations altering the structure of epidermal growth factor-like coding sequences at the Drosophila Notch locus.

The Drosophila neurogenic protein Notch is largely composed of tandemly repeated copies of an epidermal growth factor-like sequence. Notch protein contains 36 EGF-like elements, but no two are identical. In the present study, eight mutations are correlated with single amino acid substitutions in EGF-homologous elements of this protein. Genetic analyses of the mutations and comparisons of DNA from mutant and wild-type flies indicate that differentiation of function exists among the tandemly repeated EGF-like sequences and that the total number of repeats and minor variations from the consensus repeat sequence are important for wild-type Notch protein function. One group of EGF-like repeats may be involved in the formation of functional Notch dimers or multimeric proteins. EGF-like elements in a second region of the Notch protein appear to govern its interaction with the product of Enhancer of split, another neurogenic locus of D. melanogaster.

Alleles↗

PubMed2006

Regulated polyploidy in halophilic archaea.

Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1) cell lysis in agarose blocks and Southern blot analysis, and 2) Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell), and it is also downregulated (to 10 copies) as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general) than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy.

Archaea↗

PubMed1995

Sequence polymorphism in kringle IV 37 in linkage disequilibrium with the apolipoprotein (a) size polymorphism.

Apolipoprotein(a) [apo(a)] contains a variable number of identical (K-IV A/B) or nearly identical (K-IV 1, K-IV 30-37) kringle repeats that are homologous to K-IV from plasminogen. The sizes of 414 apo(a) alleles were determined by pulsed-field gel electrophoresis (PFGE) of KpnI-digested DNA. Furthermore, sequence variation in the apo(a) K-IV 30-37 domain was analysed. Reverse transcription/polymerase chain reaction (RT-PCR) cloning of human liver poly A+ RNA followed by sequencing revealed a single nucleotide exchange in the ultimate K-IV (K-IV 37) of apo(a) (codon 4168); this results in an ATG (Met) to ACG (Thr) substitution. A PCR-based restriction assay of genomic DNA demonstrated that this substitution represents a common polymorphism. In 231 unrelated Tyroleans, the frequencies for the K-IV 37 Thr and K-IV 37 Met alleles were 0.66 and 0.34, respectively. The phase between the K-IV 37 Met/Thr and the KpnI size polymorphism was determined for 224 alleles. A significant linkage disequilibrium was detected between the sequence and size polymorphisms of apo(a). K-IV 37 Met was significantly associated with KpnI allele no. 18 (DAB = 0.0267 +/- 0.0101; chi 2 = 10.09, df = 1). The Met/Thr polymorphism was further used to test whether deletions or duplications of K-IV 37 occur frequently in the apo(a) gene. Some 40 apo(a) alleles, 22 of which were from subjects that appeared to be double heterozygotes for K-IV repeat number and the Met/Thr variation were separated by PFGE and analysed for the 4168 Met/Thr polymorphism. The Met and Thr sequences were always present on different size alleles and no evidence for a duplication or deletion of K-IV 37 was obtained. This suggests that the copy number of K-IV 37 is invariable, in contrast to the highly variable K-IV A/B domain of the gene. The 4168 Met/Thr polymorphism had no effect on Lp(a) concentration, neither did it influence the lysine-binding property of the Lp(a) particle.

Adult↗

PubMed2004

Teleost fish genomes contain a diverse array of L1 retrotransposon lineages that exhibit a low copy number and high rate of turnover.

Retrotransposable elements exhibit a wide range of variation in population dynamics, abundance, and lineage diversity among host genomes across taxa. This range of diversity is illustrated by a single well-defined constituent monophyletic clade of L1 non-LTR retrotransposons that is shared between mammalian and teleost fish genomes. Despite the clear phylogenetic relationships that exist between mammalian and teleost L1 sequences, these elements exhibit markedly different dynamics within their respective taxa. While mammalian genomes typically contain a single, abundant lineage of L1 elements that traces millions of years of evolution, the zebraflsh genome was recently shown to exhibit a high diversity of ancient lineages coexisting at a very low copy number and apparently exhibiting a high rate of turnover. In the present study, a combination of degenerate PCR, lineage-specific PCR, and genomic Southern blot analysis is utilized to demonstrate high L1 lineage diversity, low copy number, and a high proportion of polymorphic inserts in the genomes of the killifish species, Fundulus heteroclitus. Additional species surveyed by degenerate PCR include Cyprinodon variegatus, Rivulus marmoratus, and Menidia beryllina. These results further support the generality of the differences that exist in host-element dynamics between teleost fish and mammalian genomes with regard to L1 retrotransposons.

Animals↗

PubMed1994

Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Amino Acid Sequence↗

PubMed1992

Isolation, characterization, and sequencing of Candida albicans repetitive element 2.

A 1059-bp Sau3A fragment, designated Candida albicans repetitive element 2 (CARE-2), was isolated from the genome of the pathogenic yeast, C. albicans. CARE-2 DNA was detected on several C. albicans chromosomes separated by transverse alternating-field electrophoresis. A high degree of interstrain variation in the pattern of hybridizing bands were observed by Southern blot analysis, with a minimum of 10-14 copies of CARE-2 per strain. A low frequency of new CARE-2 polymorphisms was observed over time for three strains grown at 25 degrees C or 37 degrees C. No new CARE-2 polymorphisms were observed from two naturally occurring switch phenotypes. To localize repeated DNA, oligodeoxyribonucleotide probes, each representing a different region of CARE-2, were hybridized to genomic blots. A lower number of copies were observed 5' and 3' to a 600-bp region of CARE-2. Nucleotide (nt) sequence analysis of CARE-2 DNA shows the element is characterized by six perfect direct repeats 6 bp in length and shows no significant DNA similarity with any known nt sequence.

Base Sequence↗