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PubMed1988

Population dynamics of movable genetic elements in Japanese natural populations of Drosophila melanogaster. I. Microevolution of male recombination determinants in experimental populations.

Five natural populations of D. melanogaster in Japan were surveyed for male recombination frequency in terms of the quantities of the mean and variance after crossing with cn bw females. The experimental condition enabled P-M system hybrid dysgenesis to take place. The means were not so different from one another, but there was a vast variation in the variances between them. Using one of these populations, two additional experimental populations were started, and the male recombination frequencies were monitored for 30 generations. Both the means and variances decreased in value at 25 degrees C. The regression line was Y = -0.000030X + 0.001706 on generation for the mean values, and was Y = -0.000098X + 0.005068 for the standard deviations in the cage population. There was no correlation between the means or the standard deviations and the copy numbers of the members of P element family as a whole. The experimental results indicate that the complete P transposable elements still retained in population were decreased in frequency and replaced by its degenerative nonautonomous forms.

Analysis of Variance↗

PubMed2003

Characterization of variants of the gene encoding the p55 antigen in Pneumocystis from rats and mice.

Variants of the p55 gene in rat-derived Pneumocystis carinii have been identified and its counterpart in mouse-derived P. carinii f. sp. muris has been cloned. By PCR amplification of P. carinii genomic DNA, five variants were identified that differed from each other in size and sequence, primarily in the number and size of encoded amino acid repeats. For P. carinii f. sp. muris, a single PCR fragment (471 bp) was obtained, which contained an incomplete ORF encoding a 157 aa protein that was most similar to a p55 variant in P. carinii, with nucleotide and amino acid sequence identity of 79 and 68 %, respectively. Southern blot analysis revealed the presence of more than one copy of the p55 gene in both Pneumocystis species. Thus, like other Pneumocystis antigens, p55 exhibits polymorphism that could potentially benefit the organism in host interactions.

Amino Acid Sequence↗

PubMed2005

Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY genes zfl1 and zfl2 on traits under selection during maize domestication.

Phenotypic variation on which selection can act during evolution may be caused by variation in activity level of developmental regulatory genes. In many cases, however, such genes affect multiple traits. This situation can lead to co-evolution of traits, or evolutionary constraint if some pleiotropic effects are detrimental. Here, we present an analysis of quantitative traits associated with gene copy number of two important maize regulatory genes, the duplicate FLORICAULA/LEAFY orthologs zfl1 and zfl2. We found statistically significant associations between several quantitative traits and copy number of both zfl genes in several maize genetic backgrounds. Despite overlap in traits associated with these duplicate genes, zfl1 showed stronger associations with flowering time, while zfl2 associated more strongly with branching and inflorescence structure traits, suggesting some divergence of function. Since zfl2 associates with quantitative variation for ear rank and also maps near a quantitative trait locus (QTL) on chromosome 2 controlling ear rank differences between maize and teosinte, we tested whether zfl2 might have been involved in the evolution of this trait using a QTL complementation test. The results suggest that zfl2 activity is important for the QTL effect, supporting zfl2 as a candidate gene for a role in morphological evolution of maize.

Biological Evolution↗

PubMed1991

Comparison of regulatory sequences and enhancer activities of SV40 variants isolated from patients with neurological diseases.

The genomic regulatory regions of human isolates of SV40 were compared by nucleotide sequencing and by biological assays of their transcriptional enhancers. SVAST is a defective genome and SVMEN an infectious genome molecularly cloned from human astrocytoma and meningioma, respectively (Krieg and Scherer, 1984). SVPML is an isolate from a case of progressive multifocal leukoencephalopathy (Weiner et al., 1972). SVAST contains a duplication of the regulatory region. One copy is virtually identical to that of SV40; the other bears a deletion of the minimal enhancer and is associated with an insertion of early/late sequences derived from the terminus of replication. The latter copy, together with its insertion, is inserted at nucleotide 4966 (SV40 number). SVMEN has an almost exact deletion of one of the 72-base-pair repeats of the natural SV40 enhancer. The regulatory sequences of SVPML were reported earlier (Martin, 1989). The enhancer activity in CV-1 cells of the lytic virus, SVPML, is 36% that of SV40. In contrast, the SVAST and SVMEN enhancers are more effective, being equal to or 64% that of SV40, respectively. The possibility that SVPML and SVAST/SVMEN originated from different variants of SV40 is discussed.

DNA, Viral↗

PubMed1998

Characterisation of a Cryptosporidium parvum-specific cDNA clone and detection of parasite DNA in mucosal scrapings of infected mice.

A cDNA library was constructed using total RNA extracted from oocysts and sporozoites of the protozoan parasite Cryptosporidium parvum. The expression library was screened with an anti-C. parvum antiserum and a clone, Cp3.4, with a 2043 bp insert, was extracted. Southern blot analysis demonstrated a single copy gene that was located on a 1.6 Mb chromosome. The gene was found to be C. parvum specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan parasites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino acids. Overall, the polypeptide has a low isoelectric point (pI) of 3.94. A total of 21 glycine/proline-rich octapeptides were identified which represented variations of a consensus sequence. The function of this protein is yet unknown. Using Cp3.4-specific PCR primers, this C. parvum gene could be amplified from as little as 0.8 pg of purified parasite DNA in a single polymerase chain reaction. Less than 0.1 ng of DNA from the ileum mucosa of immunosuppressed adult mice that had been infected with C. parvum oocysts was required to detect the parasites. In non-immunosuppressed mice that were infected and which did not shed oocysts in numbers detectable by acid-fast staining, parasite development could be detected in 25 ng of total mucosa DNA. This PCR approach may be a valuable technique for the detection of parasite infections in situations where conventional staining methods fail, such as chronic, low-grade infections or the detection of parasites in potential reservoir hosts.

Amino Acid Sequence↗

PubMed1994

Concerted evolution at the population level: pupfish HindIII satellite DNA sequences.

The canonical monomers (approximately 170 bp) of an abundant (1.9 x 10(6) copies per diploid genome) satellite DNA sequence family in the genome of Cyprinodon variegatus, a "pupfish" that ranges along the Atlantic coast from Cape Cod to central Mexico, are divergent in base sequence in 10 of 12 samples collected from natural populations. The divergence involves substitutions, deletions, and insertions, is marked in scope (mean pairwise sequence similarity = 61.6%; range = 35-95.9%), is largely confined to the 3' half of the monomer, and is not correlated with the distance among collecting sites. Repetitive cloning and direct genomic sequencing experiments failed to detect intrapopulation and intraindividual variation, suggesting high levels of sequence homogeneity within populations. The satellite sequence has therefore undergone "concerted evolution," at the level of the local population. Concerted evolution has previously almost always been discussed in terms of the divergence of species or higher taxa; its intraspecific occurrence apparently has not been reported previously. The generality of the observation is difficult to evaluate, for although satellite DNAs from a large number of organisms have been studied in detail, there appear to be little or no other data on their sequence variation in natural populations. The relationship (if any) between concerted, population level, satellite DNA divergence and the extent of gene flow/genetic isolation among conspecific natural populations remains to be established.

Animals↗

PubMed2006

PRKCA and multiple sclerosis: association in two independent populations.

Multiple sclerosis (MS) is a chronic disease of the central nervous system responsible for a large portion of neurological disabilities in young adults. Similar to what occurs in numerous complex diseases, both unknown environmental factors and genetic predisposition are required to generate MS. We ascertained a set of 63 Finnish MS families, originating from a high-risk region of the country, to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24. Initial single nucleotide polymorphism (SNP)-based association implicated PRKCA (protein kinase C alpha) gene, and this association was replicated in an independent set of 148 Finnish MS families (p = 0.0004; remaining significant after correction for multiple testing). Further, a dense set of 211 SNPs evenly covering the PRKCA gene and the flanking regions was selected from the dbSNP database and analyzed in two large, independent MS cohorts: in 211 Finnish and 554 Canadian MS families. A multipoint SNP analysis indicated linkage to PRKCA and its telomeric flanking region in both populations, and SNP haplotype and genotype combination analyses revealed an allelic variant of PRKCA, which covers the region between introns 3 and 8, to be over-represented in Finnish MS cases (odds ratio = 1.34, 95% confidence interval 1.07-1.68). A second allelic variant, covering the same region of the PRKCA gene, showed somewhat stronger evidence for association in the Canadian families (odds ratio = 1.64, 95% confidence interval 1.39-1.94). Initial functional relevance for disease predisposition was suggested by the expression analysis: The transcript levels of PRKCA showed correlation with the copy number of the Finnish and Canadian "risk" haplotypes in CD4-negative mononuclear cells of five Finnish multiplex families and in lymphoblast cell lines of 11 Centre d'Etude du Polymorphisme Humain (CEPH) individuals of European origin.

Adult↗

PubMed1991

The predicted protein sequence of a fish testis-specific alpha-tubulin cDNA shows conservation of isotype-specific amino acid substitutions.

The alpha-tubulin cDNA clone, pTUB5 (1536 bp), contains the entire coding sequence for an alpha-tubulin of 450 amino acids (mol. wt. 50,000) and is the first tubulin sequence to be reported for a fish. The transcript encoded by pTUB5 showed testis-specific expression. Its gene appears to be present at a single copy number in the trout tubulin multigene family. Comparison of the TUB5 amino acid sequence to the murine testis-specific alpha-tubulin (M alpha 3/7) revealed that eight of ten murine isotype-specific amino acid substitutions were in common. Certain of these substitutions are also conserved in other testis-specific alpha-tubulins and in alpha-tubulins of certain protozoans with flagella. The conservation of specific amino acids at these positions suggests that they may have important structural and or functional roles in the microtubules of axonemes.

Amino Acid Sequence↗

PubMed1997

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei.

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)n, (CATTA)n, (CATATC-ATTT)n and (TATTT)n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus-specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contribute to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F1 progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)53 to (TATTT)65], while the remainder of the sequence was unchanged.

Ascomycota↗

PubMed1982

Rate of turnover of structural variants in the rDNA gene family of Drosophila melanogaster.

A high degree of polymorphism for the length and copy number of rDNA spacers, in both the X and Y chromosome clusters, has been found in a wild population of Drosophila melanogaster. The genetic behaviour of rDNA structural variants in separate and mixed populations derived from isofemale lines suggest that they are not subject to strong selection and are stable for over 1,000 generations. The high structural variability suggests an evolutionary rapid process of turnover in the family which could partly explain widespread sequence homogeneity (concerted evolution) of rDNA within a species.

Animals↗

PubMed2026

Detecting and reconstructing breakage-fusion-bridge cycles from long-read sequencing using BFBArchitect.

MOTIVATION: Focal oncogene amplification is a key driver of tumor progression. Remarkably, the increased pathology depends on the context-whether the amplification is extrachromosomal (ecDNA) or intrachromosomal. EcDNA amplifications promote heterogeneity, therapy resistance, and poor prognosis. Focal intrachromosomal amplifications often arise through breakage-fusion-bridge (BFB) cycles, which produce highly rearranged but stable chromosomes. Distinguishing BFB from ecDNA remains challenging due to overlapping genomic signatures. To address this, we present BFBArchitect, a computational method leveraging long-read Oxford Nanopore data to identify BFB sequences consistent with both copy number and structural variations. RESULTS: We provide a novel combinatorial characterization of BFB, which naturally leads to an integer linear programming (ILP) optimization. The ILP optimization generates a BFB sequence that best explains experimentally observed copy numbers and foldback structural variants. We implement this idea in a tool called BFBArchitect, which achieves near-perfect accuracy in distinguishing BFB from non-BFB structures in extensive simulations as well as on 18 validated tumor samples. Moreover, it generates sequence-level BFB reconstructions that provide mechanistic insights into BFB formation, including repair mechanisms with template switching and other structural variants, and recapture of telomere for stabilization. AVAILABILITY AND IMPLEMENTATION: BFBArchitect is available at https://github.com/AmpliconSuite/BFBArchitect.

Sequence Analysis, DNA↗

PubMed2006

Structures of omega repressors bound to direct and inverted DNA repeats explain modulation of transcription.

Repressor omega regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. omega belongs to homodimeric ribbon-helix-helix (RHH2) repressors typified by a central, antiparallel beta-sheet for DNA major groove binding. Homodimeric omega2 binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5'-(A)/(T)ATCAC(A)/(T)-3', symbolized by -->) in palindromic inverted, converging (--><--) or diverging (<---->) orientation and also, unique to omega2 and contrasting other RHH2 repressors, to non-palindromic direct (-->-->) repeats. Here we investigate with crystal structures how omega2 binds specifically to heptads in minimal operators with (-->-->) and (--><--) repeats. Since the pseudo-2-fold axis relating the monomers in omega(2) passes the central C-G base pair of each heptad with approximately 0.3 A downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (-->-->), approximately 0.6 A shorter in (--><--) but would be approximately 0.6 A longer in (<---->). These variations grade interactions between adjacent omega2 and explain modulations in cooperative binding affinity of omega2 to operators with different heptad orientations.

Bacterial Proteins↗

PubMed2001

Contrasting levels of nucleotide diversity on the avian Z and W sex chromosomes.

Sex chromosomes may provide a context for studying the local effects of mutation rate on molecular evolution, since the two types of sex chromosomes are generally exposed to different mutational environments in male and female germ lines. Importantly, recent studies of some vertebrates have provided evidence for a higher mutation rate among males than among females. Thus, in birds, the Z chromosome, which spends two thirds of its time in the male germ line, is exposed to more mutations than the female-specific W chromosome. We show here that levels of nucleotide diversity are drastically higher on the avian Z chromosome than in paralogous sequences on the W chromosome. In fact, no intraspecific polymorphism whatsoever was seen in about 3.4 kb of CHD1W intron sequence from a total of >150 W chromosome copies of seven different bird species. In contrast, the amount of genetic variability in paralogous sequences on the Z chromosome was significant, with an average pairwise nucleotide diversity (d) of 0.0020 between CHD1Z introns and with 37 segregating sites in a total of 3.8 kb of Z sequence. The contrasting levels of genetic variability on the avian sex chromosomes are thus in a direction predicted from a male-biased mutation rate. However, although a low gene number, as well as some other factors, argues against background selection and/or selective sweeps shaping the genetic variability of the avian W chromosome, we cannot completely exclude selection as a contributor to the low levels of variation on the W chromosome.

Animals↗

PubMed2026

Protocol for Duplex Sequencing of Mitochondrial DNA in Single Human Oocytes.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

Humans↗

PubMed2002

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.

AT Rich Sequence↗

PubMed1996

Sporadic amplification of ID elements in rodents.

ID sequences are members of a short interspersed element (SINE) repetitive DNA family within the rodent genome. The copy number of individual ID elements varies by up to three orders of magnitude between species. This amplification has been highly sporadic in the order Rodentia and does not follow any phylogenetic trend. Using library screening and dot-blot analysis, we estimate there are 25,000 copies of ID elements in the deer mouse, 1,500 copies in the gerbil (both cricetid rodents), and 60,000 copies of either ID or ID-like elements in a sciurid rodent (squirrel). By dot-blot analysis, we estimate there are 150,000, 4,000, 1,000, and 200 copies of ID elements in the rat, mouse, hamster, and guinea pig, respectively (which is consistent with previous reports) and 200 copies in the hystricognath rodent, nutria. Therefore, a rapid amplification took place not only after the divergence of rat and mouse but also following the deer mouse (Peromyscus) and hamster split, with no evidence of increased amplifications in hystricognath rodents. No notable variations of sequences from the BC1 genes of several myomorphic rodents were observed that would possibly explain the varied levels of ID amplification. We did observe subgenera and species-group-specific variation in the ID core sequence of the BC1 gene within the genus Peromyscus. Sequence analysis of cloned ID elements in Peromyscus show most ID elements in this genus arose prior to Peromyscus subgenus divergence. Correspondence of the consensus sequence of individual ID elements in gerbil and deer mouse further confirms BC1 as a master gene in ID amplification. Several possible mechanisms responsible for the quantitative variations are explored.

Animals↗

PubMed2000

Stationary-phase variation due to transposition of novel insertion elements in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene (gum) cluster of X. oryzae pv. oryzae restored Eps production and virulence to several spv (for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, ISXo1 and ISXo2, into gumM, the last gene of the gum gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of X. oryzae pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in X. oryzae pv. oryzae.

Alleles↗

PubMed2005

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.

BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Allelic Imbalance↗