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At least 109 records · Page 6Linked to original sources
PubMed2002

A robust method for detecting CHK2/RAD53 mutations in genomic DNA.

While screening for germline CHK2 mutations in cancer cases by heteroduplex CSGE, we observed that additional PCR fragments were generated from the 3' end region of the gene that includes exons 11-14. Direct sequencing of these fragments suggested that homologous loci (possibly pseudogenes) were concomitantly being amplified. Searches of public sequence databases showed that a number of areas of the genome show a high degree of homology to exons 10-14 of the CHK2 gene. The presence of these homologous regions means that standard screening methods for detecting mutations in CHK2, based on PCR of genomic DNA, are prone to error. To circumvent this problem, we have developed a strategy, based on long-range PCR, to screen the functional copy of CHK2. Using this approach it is possible to carry out a comprehensive mutational analysis of CHK2 from genomic DNA.

Base Sequence↗
PubMed2003

[Evaluation of the effect of pollen irradiation on karyotype variability in cotton plants].

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearragements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M1 plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M1 after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.

Aneuploidy↗
PubMed1997

Inbreeding depression and inferred deleterious-mutation parameters in Daphnia.

DENG and LYNCH recently proposed a method for estimating deleterious genomic mutation parameters from changes in the mean and genetic variance of fitness traits upon inbreeding in outcrossing populations. Such observations are readily acquired in cyclical parthenogens. Selfing and life-table experiments were performed for two such Daphnia populations. We observed a significant inbreeding depression and an increase of genetic variance for all traits analyzed. DENG and LYNCH's original procedures were extended to estimate genomic mutation rate (U), mean dominance coefficient (h), mean selection coefficient (s), and scaled genomic mutational variance (Vm/Ve). On average, U, h, s and Vm/Ve (indicates an estimate) are 0.84 [corrected], 0.30, 0.14 and 4.6E-4, respectively. For the true values, the U and h are lower bounds, and s and Vm/Ve upper bounds. The present U, h and Vm/Ve are in general concordance with earlier results. The discrepancy between the present s and that from mutation-accumulation experiments in Drosophila (approximately 0.04) is discussed. It is shown that different reproductive modes do not affect gene frequency at mutation-selection equilibrium if mutational effects on fitness are multiplicative and not completely recessive.

Animals↗
PubMed1994

Evolutionary selection against change in many Alu repeat sequences interspersed through primate genomes.

Mutations have been examined in the 1500 interspersed Alu repeats of human DNA that have been sequenced and are nearly full length. There is a set of particular changes at certain positions that rarely occur (termed suppressed changes) compared to the average of identical changes of identical nucleotides in the rest of the sequence. The suppressed changes occur in positions that are clustered together in what appear to be sites for protein binding. There is a good correlation of the suppression in different positions, and therefore the joint probability of absence of mutation at many pairs of such positions is significantly higher than that expected at random. The suppression of mutation appears to result from selection that is not due to requirements for Alu sequence replication. The implication is that hundreds of thousands of Alu sequences have sequence-dependent functions in the genome that are selectively important for primates. In a few known cases Alu inserts have been adapted to function in the regulation of gene transcription.

Animals↗
PubMed1998

Inferring Deleterious-Mutation Parameters in Natural Daphnia Populations.

Deng and Lynch (1, 2) proposed to characterize deleterious genomic mutations from changes in the mean and genetic variance of fitness traits upon selfing in outcrossing populations. Such observations can be readily acquired in cyclical parthenogens. Selfing and life-table experiments were performed for two such Daphnia populations. A significant inbreeding depression and an increase of genetic variance for all traits analyzed were observed. Deng and Lynch's (2) procedures were employed to estimate the genomic mutation rate (U), mean dominance coefficient (), mean selection coefficient (), and scaled genomic mutational variance (). On average, and (^ indicates an estimate) are 0.84, 0.30, 0.14 and 4.6E-4 respectively. For the true values, the and are lower bounds, and and upper bounds.

Journal Article↗
PubMed1998

Inference of genome-wide mutation rates and distributions of mutation effects for fitness traits: a simulation study.

The properties and limitations of maximum likelihood (ML) inference of genome-wide mutation rates (U) and parameters of distributions of mutation effects are investigated. Mutation parameters are estimated from simulated experiments in which mutations randomly accumulate in inbred lines. ML produces more accurate estimates than the procedure of Bateman and Mukai and is more robust if the data do not conform to the model assumed. Unbiased ML estimates of the mutation effects distribution parameters can be obtained if a value for U can be assumed, but if U is estimated simultaneously with the distribution parameters, likelihood may increase monotonically as a function of U. If the distribution of mutation effects is leptokurtic, the number of mutation events per line is large, or if genotypic values are poorly estimated, only a lower limit for U, an upper limit for the mean mutation effect, and a lower limit for the kurtosis of the distribution can be given. It is argued that such lower (upper) limits are appropriate minima (maxima). Estimates of the mean mutational effect are unbiased but may convey little about the properties of the distribution if it is leptokurtic.

Animals↗
PubMed1997

Rapid evolution of viral RNA genomes.

Mutation rates during RNA virus replication are several orders of magnitude larger than those operating during replication of cellular DNA. This results in the continuous generation of mutant genomes and in their rating in competition with other variants present and arising in the population. The dynamic mutant distributions that constitute RNA virus populations are termed quasispecies. This concept has facilitated links between population genetics and virology and has a number of important implications for viral pathogenesis and the control of viral disease. One of them is that the mutant spectra in RNA viruses constitute large reservoirs of genetic and phenotypic variants with potentially altered biological properties. Individual mutants kept in a low proportion under a set of environmental conditions may become dominant following an environmental change. Relevant to this review are possible links between the alteration of quasispecies distributions and nutritional deficiencies and oxidative stress in cells. In addition to being a possible mechanism of viral pathogenesis, oxidative stress, and other environmental modifications resulting from nutritional imbalances, may promote population disequilibrium in replicating viruses. In particular, the increased mutagenesis mediated by oxidative DNA damage could also affect replicating RNA and integrated provirus, extending the mutant repertoire of viruses. Also, the impairment of humoral and cellular immune functions may delay or prevent viral clearance, leading to an expanded representation of viral mutants in the infected organism. Thus, nutritional deficiencies are a potential source of viral mutants with altered biological properties.

Biological Evolution↗
PubMed1999

Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay.

BACKGROUND: The goal of this study was to compare the Invader technology for the direct detection of the Factor V Leiden mutation from genomic DNA with a conventional polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. METHODS AND RESULTS: In the Invader assay, a specific upstream "invading" oligonucleotide and a partially overlapping downstream probe together form a specific structure when bound to complementary DNA template. This structure is recognized and cut at a specific site by the Cleavase enzyme, resulting in release of the 59 flap of the probe oligonucleotide. This fragment now serves as the "Invader" oligonucleotide with respect to synthetic secondary targets and secondary fluorescently labeled signal probes contained in the reaction mixture, resulting in specific cleavage of the secondary signal probe by the Cleavase enzyme. Fluorescence signal is generated when this secondary probe, labeled with dye molecules capable of fluorescence resonance energy transfer, is cleaved. Genomic DNAs isolated from peripheral blood buffy coats of patients previously tested for the Factor V Leiden mutation by PCR-RFLP were tested using an Invader assay specific for this mutation. In all 48 samples containing sufficient DNA for testing (30 normal, 16 heterozygous, 2 homozygous mutant), the genotype determined by the Invader assay was concordant with the PCR-RFLP results. CONCLUSION: These results indicate that a simple microtiter plate-based Invader assay can reliably genotype routine clinical patient samples for the Factor V Leiden point mutation without the need for PCR amplification, restriction enzyme digestion, or gel electrophoresis.

Biological Assay↗
PubMed1991

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Animals↗
PubMed2003

Identification of two novel polycystic kidney disease-1-like genes in human and mouse genomes.

Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.

Amino Acid Sequence↗
PubMed1985

The Sex-lethal gene of Drosophila: DNA alterations associated with sex-specific lethal mutations.

Genomic DNA encoding Sex-lethal, a developmental switch gene in Drosophila melanogaster that regulates sex determination and dosage compensation has been isolated. Wild-type DNA sequence organization of the gene has been compared at the restriction level with those of 17 female-specific, loss-of-function and five male-specific, gain-of-function mutant alleles. DNA lesions associated with 12 of these mutations delimit an 11 kb DNA region that is necessary for proper Sex-lethal function in females. Males who are deleted for this region are both viable and fertile. Loss-of-function alleles are associated with gross DNA alterations as well as true point mutations; the former are located throughout the region. In contrast, all five gain-of-function alleles are associated with DNA insertions that are clustered within a 1 kb portion of the Sxl gene region.

Alleles↗
PubMed2002

Modifier genes in haemophilia: their expansion in the human genome.

Mutations in factor VIII and IX genes have a determinant effect on the severity of haemophilia. Modulation of clinical manifestations depends on other genetic factors, including modifier genes. In the context of haemophilia, such genes could be the ones involved in thrombophilia. Factor V Leiden and prothrombin 20210A were studied as possible phenotypic modifiers. Inhibitor development after therapeutic factor replacement depends on the type of mutation and on the genetic factors related to the immune response of each patient. The study of all these variants in haemophiliacs constitutes an important step in prevention, prognosis and therapeutic alternatives of the disease.

Factor VIII↗
PubMed1988

Functional interactions between unlinked muscle genes within haploinsufficient regions of the Drosophila genome.

Mutations in 13 genes affecting muscle development in Drosophila have been examined in pairwise combinations for evidence of genetic interactions. Heterozygous combinations of mutations in five genes, including the gene coding for myosin heavy chain, result in more severe phenotypes than respective single heterozygous mutant controls. The various mutant interactions include examples showing allele-specific intergenic interactions, gene specific interactions, and allele-specific intragenic complementations, suggesting that some interactions result from the manner in which mutant gene products associate. Interactions that result from alterations in "+" gene copy number were also uncovered, suggesting that normal myofibril development requires that the relative amounts of respective gene products produced be tightly regulated. The importance of the latter parameter is substantiated by the finding that all five interacting loci map to disperse haploinsufficient or haplolethal regions of the genome. The implications of the present findings are discussed in relation to pursuing the phenomena involving genetic interactions to identify new genes encoding interacting myofibrillar proteins, to examine the nature of intermolecular interactions in mutant and normal development and to decipher the quantitative and temporal regulation of a large family of functionally related gene products.

Alleles↗
PubMed2002

Comprehensive scanning of the entire mitochondrial genome for mutations.

BACKGROUND: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. METHODS: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. RESULTS: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. CONCLUSIONS: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.

Child↗
PubMed1997

Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame.

It is now well recognized that mutations in the hepatitis B virus (HBV) genome occur during the natural course of chronic viral infection. Regions of the viral genome that are frequently affected by such mutations, rearrangements, and/or deletions generally involve the precore promoter, precore, and core as well as the preS gene regions. However, little is known regarding the biologic consequences of these mutations on the functional properties of the variant viral strains with respect to effects on viral replication. In this study, we investigated the functional significance of precore promoter and precore gene mutations that reduce or abolish the synthesis of hepatitis B e antigen (HBeAg). We found that precore promoter mutations diminished the expression of HBeAg but did not affect the synthesis of pregenomic RNA. However, these precore mutations were associated with a modest increase in HBV replication. In contrast, a naturally occurring mutant that carries a termination codon in position 28 of the precore open reading frame demonstrated increased encapsidation of pregenomic mRNA into nucleocapsid particles. Consequently, this variant viral strain demonstrated a substantial increase in the level of viral replication compared to "wild-type" HBV and other precore promoter mutant viral strains. These studies suggest that substitutions in the precore promoter and precore gene not only alter the synthesis of HBeAg but also affect the level of viral replication.

Codon, Terminator↗
PubMed2006

Analysis of Toll-like receptor 2, 4, 6 and 9 genome DNA mutations in patients with tractable and intractable gastric mucosal diseases.

The possible involvement of Toll-like receptor (TLR) genome DNA in the prolongation and relapse of inflammatory intestinal diseases and alcoholic hepatic diseases has been reported. In this study, we examined the relationship of mutations of the TLR 2, 4, 6 and 9 genomic DNA to recurrent or intractable gastritis or gastric ulcers. The subjects were 32 patients, including 6 with H. pylori (Hp)-positive gastritis, 4 with Hp-negative gastritis, 10 with Hp-positive tractable gastric ulcer, 5 with Hp-positive recurrent gastric ulcer after Hp eradication, and 7 with Hp-negative easily recurrent gastric ulcer after Hp eradication. Gastric mucosal tissue and peripheral blood specimens were collected from each of the patients. DNA was extracted from the tissue and blood specimens and subjected to electrophoresis by the PCR method, using the oligonucleotide primers of TLR 2, 4, 6 and 9. The gastric mucosal tissue specimens were collected endoscopically from the sites of the lesions. Subsequently, the presence or absence of genomic DNA mutations in the blood and tissue specimens was examined using a DNA sequencer. TLR 2, 4, 6 or 9 DNA mutations were not observed in any of the gastric mucosal or peripheral blood specimens obtained from patients with tractable gastritis or gastric ulcer, or from those with intractable gastric ulcer who were Hp-positive or Hp-negative or had become Hp-negative after eradication therapy. These data suggest that mutations of the TLR 2, 4, 6 and 9 genome DNA may not be involved in the recurrence, delayed healing or intractability of gastritis and gastric ulcers.

Base Sequence↗
PubMed2007

Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila.

Spontaneous mutations are the source of genetic variation required for evolutionary change, and are therefore important for many aspects of evolutionary biology. For example, the divergence between taxa at neutrally evolving sites in the genome is proportional to the per nucleotide mutation rate, u (ref. 1), and this can be used to date speciation events by assuming a molecular clock. The overall rate of occurrence of deleterious mutations in the genome each generation (U) appears in theories of nucleotide divergence and polymorphism, the evolution of sex and recombination, and the evolutionary consequences of inbreeding. However, estimates of U based on changes in allozymes or DNA sequences and fitness traits are discordant. Here we directly estimate u in Drosophila melanogaster by scanning 20 million bases of DNA from three sets of mutation accumulation lines by using denaturing high-performance liquid chromatography. From 37 mutation events that we detected, we obtained a mean estimate for u of 8.4 x 10(-9) per generation. Moreover, we detected significant heterogeneity in u among the three mutation-accumulation-line genotypes. By multiplying u by an estimate of the fraction of mutations that are deleterious in natural populations of Drosophila, we estimate that U is 1.2 per diploid genome. This high rate suggests that selection against deleterious mutations may have a key role in explaining patterns of genetic variation in the genome, and help to maintain recombination and sexual reproduction.

Animals↗