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PubMed2005

Detection of DNA copy number alterations using penalized least squares regression.

MOTIVATION: Genomic DNA copy number alterations are characteristic of many human diseases including cancer. Various techniques and platforms have been proposed to allow researchers to partition the whole genome into segments where copy numbers change between contiguous segments, and subsequently to quantify DNA copy number alterations. In this paper, we incorporate the spatial dependence of DNA copy number data into a regression model and formalize the detection of DNA copy number alterations as a penalized least squares regression problem. In addition, we use a stationary bootstrap approach to estimate the statistical significance and false discovery rate. RESULTS: The proposed method is studied by simulations and illustrated by an application to an extensively analyzed dataset in the literature. The results show that the proposed method can correctly detect the numbers and locations of the true breakpoints while appropriately controlling the false positives. AVAILABILITY: http://bioinformatics.med.yale.edu/DNACopyNumber CONTACT: hongyu.zhao@yale.edu SUPPLEMENTARY INFORMATION: http://bioinformatics.med.yale.edu/DNACopyNumber.

Algorithms↗

PubMed1999

Use of pulsed-field gel electrophoresis for molecular epidemiologic and population genetic studies of Mycobacterium tuberculosis.

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology of Mycobacterium tuberculosis. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of M. tuberculosis were genotyped with four different PFGE enzymes (AseI, DraI, SpeI, and XbaI), as well as by RFLP analysis with IS6110. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS6110 typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS6110 and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping is dependent upon the number of IS6110 elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that M. tuberculosis is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of M. tuberculosis.

DNA Transposable Elements↗

PubMed2002

Allopolyploidization and evolution of species with reduced floral structures in Lepidium L. (Brassicaceae).

Understanding the pattern of speciation in a group of plants is critical for understanding its morphological evolution. Lepidium is the genus with the largest variation in floral structure in Brassicaceae, a family in which the floral ground plan is remarkably stable. However, flowers in more than half of Lepidium species have reduced stamen numbers, and most of these also have reduced petals. The species with reduced flowers are geographically biased, distributed mostly in the Americas and Australia New Zealand. Previous phylogenetic studies using noncoding regions of chloroplast DNA and rDNA internal transcribed spacer were incongruent in most New World species relationships. These data, combined with the presence of many polyploid Lepidium species, implied a reticulate history of the genus but did not provide enough information to infer the evolutionary pattern of flower structures. To address this question more thoroughly, sequences of the first intron of a single copy nuclear gene, PISTILLATA, were determined from 43 species. Phylogenetic analysis of the PI intron suggests that many species in the New World have originated from allopolyploidization, and that this is correlated with floral reduction. Interspecific hybrids were generated to understand why allopolyploidization is associated with reduced flowers. The phenotypes of F1 flowers indicate allelic dominance of the absence of lateral stamens, suggesting that propagation of dominant alleles through interspecific hybridization could account for the abundance of the allopolyploid species without lateral stamens.

Base Sequence↗

PubMed1992

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.

Animals↗

PubMed1981

Contextural genomic rearrangements of variable-antigen genes in Trypanosoma brucei.

We have described some rearrangements of a variable-antigen gene in T. brucei. We suggest that there are two copies of the ILTat 1.2 variable-antigen gene in each of a number of trypanosome clones closely related by sequential relapses. Both copies of the gene undergo rearrangements, apparently the result of insertion and deletion of lengths of DNA in a region at or beyond the 3' end of the coding sequence, giving rise to different-size restriction enzyme fragments in different clones of trypanosomes. No feature of these rearrangements can be correlated with expression of the gene. Our data differ from those of Hoeijmakers et al. (1980) in two important respects: (1) Neither copy of the gene remains in a constant genomic context in all trypanosome clones. (2) We do not see an extra copy associated with the expression of the gene. These observations do not suggest any obvious mechanism for the phenomenon of antigenic variation.

Animals↗

PubMed1984

Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation.

The entire subtilisin structural gene from Bacillus subtilis I168 has been cloned, and its nucleotide sequence has been determined. When expressed on a high-copy-number shuttle vector, a fivefold increase in serine protease activity was observed. The DNA sequence of the gene is 80% homologous to the Bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin BPN'. The chloramphenicol resistance determinant of a plasmid integrated at the subtilisin locus was mapped by PBS1 transduction and was found to be linked to glyB (83%) and argC (60%), but not with metC or purB . The chromosomal locus containing the wild-type subtilisin allele was replaced with an in vitro-derived allele of the gene (delta apr-684) that contained a 684-base-pair deletion. The technique used for introducing the deletion is a variation of the gene replacement methods used in Saccharomyces cerevisiae and Escherichia coli. When used in B. subtilis, deletion mutants could be directly screened among the transformants. Physiological characterization of the delta apr-684 mutation revealed no discernable effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity. A model is presented that outlines the pathway for plasmid integration and deletion formation in B. subtilis.

Bacillus subtilis↗

PubMed1992

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.

Animals↗

PubMed2005

Retrotransposon characterisation and fingerprinting of apple clones by S-SAP markers.

Retrotransposons have been found to comprise the most common class of transposable elements in eukaryotes and to occur in high copy number in plant genomes. Several of these elements have been sequenced and were found to display a high degree of heterogeneity and insertional polymorphism, both within and between species. The dispersion, ubiquity and prevalence of retrotransposons in plant genomes provide an excellent basis for the development of marker systems and, hence, may be good molecular candidates in distinguishing among apple clones, when they represent bud mutations of the original variety, considering that the random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) used thus far in fingerprinting analyses have failed to meet discrimination expectations. The technique called sequence-specific amplified polymorphism (S-SAP), which makes it possible to identify dominant markers for the detection of variation in the DNA flanking the retrotransposon insertion site, was used in the present study to distinguish several clones of the cultivars 'Gala' and 'Braeburn' in apple fingerprinting. Moreover, our results suggest that the bud mutations, which have generated new patented varieties of 'Gala' and 'Braeburn', appear to derive from retrotransposon insertion.

Alleles↗

PubMed2005

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags.

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.

Alleles↗

PubMed2000

Typing of Candida glabrata in clinical isolates by comparative sequence analysis of the cytochrome c oxidase subunit 2 gene distinguishes two clusters of strains associated with geographical sequence polymorphisms.

We tested whether comparative sequence analysis of the mitochondrion-encoded cytochrome c oxidase subunit 2 gene (COX2) could be used to distinguish intraspecific variants of Candida glabrata. Mitochondrial genes are suitable for investigation of close phylogenetic relationships because they evolve much faster than nuclear genes, which in general exhibit very limited intraspecific variation. For this survey we used 11 clinical isolates of C. glabrata from three different geographical locations in Brazil, 10 isolates from one location in the United States, 1 American Type Culture Collection strain as an internal control, and the published sequence of strain CBS 138. The complete coding region of COX2 was amplified from total cellular DNA, and both strands were sequenced twice for each strain. These sequences were aligned with published sequences from other fungi, and the numbers of substitutions and phylogenetic relationships were determined. Typing of these strains was done by using 17 substitutions, with 8 being nonsynonymous and 9 being synonymous. Also, cDNAs made from purified mitochondrial polyadenylated RNA were sequenced to confirm that our sequences correspond to the expressed copies and not nuclear pseudogenes and that a frameshift mutation exists in the 3' end of the coding region (position 673) relative to the Saccharomyces cerevisiae sequence and the previously published C. glabrata sequence. We estimated the average evolutionary rate of COX2 to be 11.4% sequence divergence/10(8) years and that phylogenetic relationships of yeasts based on these sequences are consistent with rRNA sequence data. Our analysis of COX2 sequences enables typing of C. glabrata strains based on 13 haplotypes and suggests that positions 51 and 519 indicate a geographical polymorphism that discriminates strains isolated in the United States and strains isolated in Brazil. This provides for the first time a means of typing of Candida strains that cause infections by use of direct sequence comparisons and the associated divergence estimates.

Bacterial Proteins↗

PubMed2002

Phylogeny of Gaertnera Lam. (Rubiaceae) based on multiple DNA markers: evidence of a rapid radiation in a widespread, morphologically diverse genus.

Phylogenetic relationships among 28 of the 68 species of the paleotropical genus Gaertnera (Rubiaceae) and two related genera were inferred from nucleotide sequence variation in four nuclear DNA (nDNA) markers: the internal transcribed spacers of nuclear rDNA (ITS), the large and small copies of phosphoenolpyruvate carboxylase (PepC-large and PepC-small), and triose phosphate isomerase (Tpi). Phylogenetic analysis of the combined nDNA dataset suggested that Gaertnera is monophyletic, but genetic variation among species was insufficient to reconstruct well-supported relationships within the genus. This was counter to expectations based on the very distinct morphologies and widespread distribution of the genus (West Africa to Sulawesi). Molecular clock analyses suggested variable dates of origin for Gaertnera depending upon the calibration method used. The most plausible calibration implies that Gaertnera migrated to Africa during the early Tertiary, possibly via a boreotropical land bridge and suggests that Gaertnera started to radiate 5.21 +/- 0.14 million years ago. This implies that range expansion in the group has occurred via a number of long-distance dispersal events rather than vicariance. The molecular clock estimate in turn estimated an unusually rapid lineage diversification rate within the radiation of 0.717-0.832 species/million years, comparable to those estimated for radiations on oceanic islands. Although low interspecific competition levels may have contributed to the diversification of Gaertnera on Mauritius, the mechanisms driving the rapid radiation of the group in other parts of its range remain elusive.

Base Sequence↗

PubMed2005

Evolutionary mechanisms shaping the genomic structure of the Williams-Beuren syndrome chromosomal region at human 7q11.23.

About 5% of the human genome consists of segmental duplications or low-copy repeats, which are large, highly homologous (>95%) fragments of sequence. It has been estimated that these segmental duplications emerged during the past approximately 35 million years (Myr) of human evolution and that they correlate with chromosomal rearrangements. Williams-Beuren syndrome (WBS) is a segmental aneusomy syndrome that is the result of a frequent de novo deletion at 7q11.23, mediated by large (approximately 400-kb) region-specific complex segmental duplications composed of different blocks. We have precisely defined the structure of the segmental duplications on human 7q11.23 and characterized the copy number and structure of the orthologous regions in other primates (macaque, orangutan, gorilla, and chimpanzee). Our data indicate a recent origin and rapid evolution of the 7q11.23 segmental duplications, starting before the diversification of hominoids (approximately 12-16 million years ago [Mya]), with species-specific duplications and intrachromosomal rearrangements that lead to significant differences among those genomes. Alu sequences are located at most edges of the large hominoid-specific segmental duplications, suggesting that they might have facilitated evolutionary rearrangements. We propose a mechanistic model based on Alu-mediated duplicated transposition along with nonallelic homologous recombination for the generation and local expansion of the segmental duplications. The extraordinary rate of evolutionary turnover of this region, rich in segmental duplications, results in important genomic variation among hominoid species, which could be of functional relevance and predispose to disease.

Alu Elements↗

PubMed2004

Error-prone replication for better or worse.

Precise genome duplication requires accurate copying by DNA polymerases and the elimination of occasional mistakes by proofreading exonucleases and mismatch repair enzymes. The commonly held belief that 'if something is worth doing, then it's worth doing well' normally applies to DNA replication and repair, however, there are exceptions. This review describes elements that are crucial to cell fitness, evolution and survival in the recently discovered error-prone DNA polymerases. Large numbers of errant DNA polymerases, spanning microorganisms to humans, are used to rescue stalled replication forks by copying damaged DNA and even undamaged DNA to generate 'purposeful' mutations that generate genetic diversity in times of stress. Here we focus on low-fidelity polymerases from bacteria, comparing Escherichia coli, archeabacteria and those most recently discovered in Gram-positive Bacilli, Streptococcus, pathogenic Mycobacterium and intein-containing cyanobacteria.

Adaptation, Biological↗

PubMed2003

Variability of HXT2 at the protein and gene level among the Saccharomyces sensu stricto group.

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.

Chromosomes, Fungal↗

PubMed1992

A fourth family of the Plasmodium falciparum S-antigen.

The S-antigen of Plasmodium falciparum is a highly diverse heat stable protein that is located in the parasitophorous vacuole of the mature asexual intraerythrocytic parasite. The gene for S-antigen exists within the parasite population as multiple alleles at a single locus. Its sequence contains a large central block of tandemly arranged peptides that are identical or very similar in one allele but differ widely in sequence, repeat length and number among different alleles, and consequently antigenic specificity. Thus, antibodies directed against the repeat region can be used to define the serotype of an S-antigen. Flanking this repeat block are 2 short regions of non-repetitive sequence which have been described as occurring in three different forms, each of which is used to define a single S-antigen family. We present the S-antigen sequence for the isolate 3D7 which defines not only a novel serotype but also a novel S-antigen family. The central repeat block is composed of 57 copies of an 8-residue peptide with consensus sequence ED(E/K)VSNG(R/G). Comparison of the four S-antigen families reveals that they differ considerably from each other with variation being most pronounced in the carboxy terminal-flanking region. This pattern of sequence variation differs considerably from that found for MSA-1 and MSA-2, the only other diverse proteins of P. falciparum for which sequence information is available.

Amino Acid Sequence↗

PubMed1992

Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat.

A method of restriction fragment length polymorphism (RFLP) analysis was used to estimate the amount of genetic divergence between the spontaneously hypertensive rat (SHR) strain and the Wistar-Kyoto (WKY) strain. DNA from each strain was digested with eight restriction endonucleases and hybridized with six single copy gene sequences. The number of hybridization bands in each digestion was used to estimate the total number of bases analyzed and RFLPs were scored as single mutations. Divergence was then estimated by dividing the number of mutations by the number of bases analyzed. In a total of 808 bases analyzed in WKY rats, a minimum of 13 mutations were scored in SHR, which yields a nucleotide divergence of 1 change per 62 bp. This is an extremely high amount of divergence given the known origin of these two strains and is comparable to the maximum divergence possible between unrelated humans.

Animals↗

PubMed1991

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector.

Phe82 is the most studied invariant residue of cytochrome c. However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e. using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector. Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level. Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon. The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast. All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c. However the F82C, F82P, and F82R variants grow at a significantly lower rate. After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified. It is concluded that all 20 amino acids are capable of supporting function. Reasons for the evolutionary invariance of Phe82 are discussed.

Alleles↗

PubMed1986

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Animals↗