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PubMed1985

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. I. Isolation and biochemical genetic characterization.

Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase alpha was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent Km for dCTP and the apparent Ki for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.

Animals↗
PubMed2004

A genomewide search using an original pairwise sampling approach for large genealogies identifies a new locus for total and low-density lipoprotein cholesterol in two genetically differentiated isolates of Sardinia.

A powerful approach to mapping the genes for complex traits is to study isolated founder populations, in which genetic heterogeneity and environmental noise are likely to be reduced and in which extended genealogical data are often available. Using graph theory, we applied an approach that involved sampling from the large number of pairwise relationships present in an extended genealogy to reconstruct sets of subpedigrees that maximize the useful information for linkage mapping while minimizing calculation burden. We investigated, through simulation, the properties of the different sets in terms of bias in identity-by-descent (IBD) estimation and power decrease under various genetic models. We applied this approach to a small isolated population from Sardinia, the village of Talana, consisting of a unique large and complex pedigree, and performed a genomewide search through variance-components linkage analysis for serum lipid levels. We identified a region of significant linkage on chromosome 2 for total serum cholesterol and low-density lipoprotein (LDL) cholesterol. Through higher-density mapping, we obtained an increased linkage for both traits on 2q21.2-q24.1, with a LOD score of 4.3 for total serum cholesterol and of 3.9 for LDL cholesterol. A replication study was performed in an independent and larger set from a genetically differentiated isolated population of the same region of Sardinia, the village of Perdasdefogu. We obtained consistent linkage to the region for total serum cholesterol (LOD score 1.4) and LDL cholesterol (LOD score 2.2), with a level of concordance uncommon for complex traits, and refined the location of the quantitative-trait locus. Interestingly, the 2q21.1-22 region has also been linked to premature coronary heart disease in Finns, and, in the adjacent 2q14 region, significant linkage with triglycerides has been reported in Hutterites.

Cholesterol↗
PubMed1993

Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo II alpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo II alpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.

Antineoplastic Agents↗
PubMed1988

Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations.

Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae. Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7). New alleles of the CDC2 and CDC17 genes were recovered among these mutants. The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity. Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination. These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally. Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.

Cell Cycle↗
PubMed2006

Frequency of the thiopurine S-methyltransferase alleles in the ancient genetic population isolate of Sardinia.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an enzyme involved in the normal metabolic inactivation of thiopurine drugs. Patients with intermediate or no TPMT activity are at risk of toxicity after receiving standard doses of thiopurine drugs and it was shown that inter-individual differences in response to these drugs is largely determined by genetic variation at the TPMT locus. OBJECTIVE: This study was designed to investigate in the Sardinian population the frequency distribution of four of the most common variants accounting for TPMT deficiency and to conduct comparative analyses with other populations in order to obtain insights into the main factors that have shaped diversity at the TPMT locus in Sardinia. METHODS: DNA was extracted in 259 Sardinians and the frequencies of allelic variants of TPMT were determined using polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Among the 259 Sardinians genotyped, 6.95% were found to be heterozygous for one of four TPMT variants screened; for each variant the frequency estimate was 1.74%, 0.58%, 0.39% and 0.77% for TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C respectively. CONCLUSIONS: Although Sardinia does not show reduced diversity at the TPMT locus, the spectrum of TPMT allele frequencies affords evidence of remarkable influence of genetic drift and founder effects throughout its population history. In the broad context of the European TPMT diversity, the Sardinians come out as outliers, an observation consistent with previous genetic inferences that Sardinia has features of a genetic isolate.

Adult↗
PubMed1999

Mating type-correlated molecular markers and demonstration of heterokaryosis in the phytopathogenic fungus Thanatephorus cucumeris (Rhizoctonia solani) AG 1-IC by AFLP DNA fingerprinting analysis.

The destructive soil-borne plant pathogenic basidiomycetous fungus Thanatephorus cucumeris (Frank) Donk [anamorph: Rhizoctonia solani Kühn] is not a homogeneous species, but is composed of at least twelve anastomosis groups (AG), which seem to be genetically isolated. The genetics of several T. cucumeris anastomosis groups has been studied by analysis of heterokaryotic tuft formation in the area of contact between homokaryotic single-spore isolates, revealing that AG 1 is heterokaryotic and bipolar. To prove that tuft formation is due to heterokaryosis, AFLP DNA fingerprinting has been applied to a heterokaryotic T. cucumeris AG 1-IC isolate, its homokaryotic single spore-derived progeny, and newly formed heterokaryons. By means of AFLP markers, it is demonstrated that fluffy tufts formed upon pairing of homokaryons from different mating types are newly formed heterokaryons. Mating type-correlated markers have also been found, which will be useful for future studies of the genetics of this fungal species complex.

DNA Fingerprinting↗
PubMed2006

Disassociation between weak sexual isolation and genetic divergence in a hermaphroditic land snail and implications about chirality.

Examination of the association between reproductive isolation and genetic divergence in a variety of organisms is essential for elucidating the mechanisms causing speciation. However, such studies are lacking for hermaphrodites. We measured premating (sexual) isolation in species pairs of the hermaphroditic land snail Albinaria and we compared it with their genetic divergence. We did not find substantial sexual isolation barriers between the species studied. The absence of strong sexual isolation between species implies its minor effect in the evolution of this genus, because distributional, population and life-history characteristics of Albinaria make mate-choice possibly redundant. Furthermore, we found disassociation between genetic divergence and sexual isolation, suggesting that they do not form necessarily a cause-effect duet. However, Albinaria voithii, the only dextral Albinaria species, shows strong sexual isolation against the other sinistral species. We discuss whether change in coiling either has triggered instantaneous speciation, or is an example of character displacement.

Animals↗
PubMed2004

[Genetic and taxonomic diversity of the house mouse Mus musculus from the asian part of the former Soviet Union].

Genetic diversity of the house mouse Mus musculus from 12 local populations (n = 65) of the central and eastern parts of the former Soviet Union was examined using RAPD-PCR. About 400 loci were identified, encompassing approximately 500 kb of the mouse genome. Genetic diversity was assessed using NTSYS, POPGENE, TFPGA, and TREECON software programs. In general, the house mouse sample from the regions examined was characterized by moderate genetic variation: polymorphism P = 95.6%, P99 = 60.7%, P95 = 24.2%; heterozygosity H = 0.089; the mean observed number of alleles n(a) = 1.97; effective number of alleles n = 1.13; intrapopulation differentiation deltaS = 0.387; gene diversity h = 0.09. Individual local populations displayed different levels of genetic isolation: the genetic subdivision index G(st) varied from 0.086 to 0.324 at gene flow Nm varying from 5.3 to 1.05, while the interpopulation genetic distance D(N) ranged from 0.059 to 0.186. Most of the genetic diversity of the total sample resided within the local populations: H(S) = 0.6, total gene diversity H(T) = 0.09. The exact test for differentiation, however, did not confirm the affiliation of all the mice examined to one population: chi2 = 1446, d.f. = 724, P = 0.000. Molecular markers specific to four subspecies (musculus, castaneus, gansuensis, and wagneri) were identified. Moreover, in some cases the populations and individual animals exhibited traits of different subspecies, suggesting their introgressive hybridization. It was demonstrated that the house mouse fauna on the territories investigated was characterized by the prevalence of musculus-specific markers, while gansuensis-specific markers ranked second. The castaneus-specific markers were highly frequent in the Far East, but almost absent in Central Asia, where wagneri-specific markers were detected. It was suggested that house mice from Turkmenistan could belong to one of the southern subspecies, which had not deeply penetrated into the Asian fauna of the former Soviet Union. In phenogenetic (UPGMA) and phylogenetic (NJ) reconstructions this form with the high bootstrap support was placed at the tree base, while the isolation of other clusters was not statistically significant. It is thus likely that the house mice from Turkmenistan are closest to the ancestral form of the genus Mus on the territory of the former Soviet Union.

Animals↗
PubMed2001

The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis.

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec(-) mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37 degrees C. Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.

Cell Division↗
PubMed1980

[Genetic control of pathways of pyrimidine metabolism in Aspergillus nidulans. 1. Isolation and genetic analysis of 6-azauracil resistant mutants].

147 mutants exhibiting the resistance to toxic effect of 6 azauracil have been isolated by nitrosoguanidine treatment from the wild strain of Aspergillus nidulans. This mutants have been divided into 11 phenotypic groups according to its cross-resistance to 5-fluoroderivatives of uracil, uridine and deoxyuridine. The genetic analysis has shown that all mutations of resistance are of nuclear origin and dominant nature, and they are distributed on six loci of the chromosome VIII.

Aspergillus nidulans↗
PubMed2006

Genetic study of Sardinian patients with Alzheimer's disease.

We describe the genetic analysis of an Alzheimer's disease (AD) sample derived from a genetically isolated population. Genetic assessment included the analysis of genes involved in AD, such as the genes for amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). We also assessed genes for some proteins that constitute the gamma-secretase complex: nicastrin (NCSTN), presenilin enhancer-2 (PEN2), in addition to the AD risk factor apolipoprotein E (APOE). Using polymerase chain reaction and single strand conformational polymorphism method, screens for APP, PSEN1 and PSEN2 genes revealed one mutation in PSEN1. Furthermore, we found an intronic +17G>C polymorphism in PEN2 which, in homozygous form, was greater in early onset Alzheimer's disease (EOAD) compared to controls, and one haplotype in the NCSTN gene which was linked to EOAD and familial AD (FAD). Finally, the genotyping of APOE confirmed that the varepsilon4 allele could be a risk factor for the onset of AD, in particular for FAD subjects. In conclusion, these results show the existence of Sardinian genetic peculiarities, essential in studies regarding genetically inherited and multifactorial disorders, as AD.

Aged↗
PubMed2002

HLA polymorphism in a Majorcan population of Jewish descent: comparison with Majorca, Minorca, Ibiza (Balearic Islands) and other Jewish communities.

'Chueta' was the name given to the Catholic descendants of Jewish victims of the last Spanish Inquisition process in Majorca Island in the western Mediterranean. We have studied the allele distribution of HLA-A, -B, -Cw, -DRB1 and -DQB1 loci of 103 random, healthy, unrelated individuals belonging to the ancient Majorcan Jewish community, known locally as Chuetas, and 589 individuals from the Balearic population selected because of their typical Balearic - Majorca, Minorca or Ibiza - lineages and according to their ancestor's place of birth. Our aim was to establish the genetic relationship between Majorcan Chuetas, and Balearic and other Jewish and Mediterranean populations. Our results have shown that, to a remarkable extent, they have retained their biological identity, with a unique pattern, in terms of gene and haplotype frequencies, separate from the other populations of Majorca. The Chuetas were found to be more related to Moroccan and Libyan Jews than other Majorcans. Characteristic Jewish haplotypes, A26-B38-DRB1*13, A24-B38-DRB1*11, A1-B52-DRB1*15/16, were found in our study. Some peculiarities were observed in the distribution of common haplotypes among the three main Balearic Islands. The Ibizan population was genetically different from the other Balearic populations, with a high frequency of some haplotypes, for example, A29-Cw*16-B44-DRB1*07-DQB1*03; A1-Cw*07-B8-DRB1*03-DQB1*02. We also found a new haplotype, A25-Cw*12-B39-DRB1*11-DQB1*03(3.5%), in Ibizans and a more limited variability in the HLA alleles that were expressed, perhaps because of genetic isolation. The genetic diversity of the populations from Majorca and Minorca were similar and more related to the mainland Spanish population.

Alleles↗
PubMed1998

Isolation and genetic characterization of the porcine apolipoprotein E gene.

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (apo-E) gene. A single positive recombinant phage clone containing a 10.7-kb insert was isolated from a porcine genomic library, and a 4.2-kb fragment was subcloned and sequenced. The 4.2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)13 microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2.1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.

Animals↗
PubMed2000

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

Agrobacterium tumefaciens↗
PubMed2003

Striking differentiation of sub-populations within a genetically homogeneous isolate (Ogliastra) in Sardinia as revealed by mtDNA analysis.

Since the reduced genetic diversity found in isolates should simplify the study of complex traits, analyses of patterns of homogeneity within populations are of particular interest. We analysed the mtDNA haplogroups and hypervariable segment I (HVS-I) sequences of 475 individuals from a geographically restricted and isolated area (Ogliastra) within Sardinia, comprehending 175 random samples from 20 out of 23 villages. The remaining 300 subjects were chosen from the other three villages, Talana, Urzulei and Perdasdefogu, by sampling all maternal lineages. A comparison with other European populations reveals that Ogliastra ranks among the most genetically homogenous population and that it has been small and isolated throughout its history. The lack of variation and the high genetic homogeneity indicate that an important founder event and a demographic expansion took place during the Neolithic (approximately 7700 years before present) in Ogliastra's mtDNA gene pool. We present highly resolved phylogenetic networks for Ogliastra and for the three sub-isolates. MtDNA differentiation in the sub-populations versus Ogliastra is revealed by a strong demarcation in their genetic pools due to distinctive founder effects and genetic drift. We found that genetic homogeneity strictly depends on a scale factor in population size and on sampling methodology. The outstanding homogeneity and the reduced female gene pool observed in Ogliastra, in the European context, hide an extremely marked differentiation in sub-isolates originated from the same archaic population. Although Ogliastra can be considered a genetically homogeneous isolate, small villages' divergent genetic histories underline the importance of more systematic analysis of DNA variation between and within populations.

DNA↗
PubMed2006

Heterozygosities of 735 microsatellite markers and background linkage disequilibrium in the Korean population.

Suitability of a specific population for linkage disequilibrium mapping studies of complex traits may be assessed by investigating the background linkage disequilibrium (BLD). We are unaware of studies for quantifying the degree of BLD in the Korean population, although the population may be a good candidate for mapping of complex trait genes through whole-genome association studies. It is useful to investigate the properties of genetic isolates in East Asia and to compare them to genetic isolates in Europe. We analyzed the extent of BLD in the Korean population using 735 microsatellite markers and compared the results with the Icelander population, which is one of the European expanded genetic isolates. The Korean population exhibited a level of BLD comparable with the Icelander population. The inference of population structure using the model with admixture showed that each individual has allele copies originating from K populations in equal proportions. Therefore, we believe that factors other than genetic distance, such as recent admixture, have not contributed to the level of BLD. Our results showed that the Korean population, which is an expanded population with no evidence of admixture, has a BLD level comparable with the Icelander population. Therefore, the Korean population can be used for fine mapping of either complex traits or monogenic diseases.

Adult↗
PubMed2006

Differences in the API 20E biochemical patterns of clinical and environmental Vibrio parahaemolyticus isolates.

Genetic differences in clinical and environmental strains of Vibrio parahaemolyticus have been widely used as criteria in identifying pathogenic isolates. However, few studies have been carried out to assess the differences in biochemical characteristics of V. parahaemolyticus isolates from human and environmental sources. We compared the biochemical profiles obtained by the characterization of V. parahaemolyticus isolates from human infections and the marine environment using the API 20E system. Environmental and clinical isolates showed significant differences in the gelatin and arabinose tests. Additionally, clinical isolates were correctly identified according to the API 20E profile using 0.85% NaCl diluent, but they presented nonspecific profiles with 2% NaCl diluent. In contrast, use of 2% NaCl diluent facilitated correct identification of the environmental isolates. Clinical isolates showed significant differences in up to five biochemical tests with respect to the API 20E database. The API 20E system is widely used in routine identification of bacteria in clinical laboratories, and this discrepancy in an important number of biochemical tests may lead to misidentification of V. parahaemolyticus infection.

Animals↗
PubMed1994

Molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V. cholerae isolates.

Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V. cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993). The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S. Gulf Coast, and 5 O139 Bengal isolates. Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates. O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites. The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide. Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.

Bacterial Typing Techniques↗