Host-mediated assay with yeast and rats using probenecid (Benemid®) to block the renal tubular excretion of cyclophosphamide metabolites

The genetic activity of cyclophosphamide (Cy) was tested in the host-mediated assay (injection of yeasts into the peritoneal cavity of rats) modified by the use of probenecid (Pro) (Benemid®) to block the renal tubular excretion of the genetically active metabolites. The genetic test system used was the induction of mitotic gene conversion in two unlinked loci of a diploid strain of Saccharomyces cerevisiae. By this method the genetic effect of Cy was doubled in comparison with the case of administering Cy alone. Compared with the animals which received only Pro, increases of conversion frequencies of 20 times in the ade2 locus and of 15 times in the trp5 locus were found.     

Cyclophosphamide: pilot study of genetically active metabolites in the urine of a treated human patient. Induction of mitotic gene conversions in yeast

(1) From a female patient who received four successive infusions of cyclophosphamide (Endoxan; 10, 15, 30 mg/kg), nine samples of urine were collected at different intervals from the beginning of the infusions. (2) Diploid yeast cells, heteroallelic in two gene loci, were treated with the urine, and the induction of mitotic gene conversions (intragenic recombinations) was investigated. (3) Urine, collected before treatment and diluted 3:1 by phosphate buffer (pH 7.0), produced the same control value as the buffer alone. (4) The most reliable conversion frequencies from among undiluted and 1:1 or 3:1 diluted urine were obtained with the urine diluted 3:1. (5) The first rise in conversion frequency was observed 2 h after the start of the infusion; maximal values appeared 5–6 h later. 23 h after the start, the frequency of convertants was low again, but much higher than the statistically relevant limit. (6) The frequency of induced convertants seemed to depend not only on the dose and the interval between infusion and collection of the urine but also on the duration of the infusion. (7) The pilot experiments clearly demonstrate the utility of the yeast conversion test for the determination of the amount of genetically active metabolites in the urine. Moreover, the test also gives statistically relevant results with low doses of drugs used in cancer therapy.     

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (<10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.     

Association of Disomic Chromosome Loss with Ems-Induced Conversion in Yeast

Experimental tests with the yeast Saccharomyces cerevisiae of a previously proposed model suggesting a causal relationship between disomic chromosome loss (n + 1 → n) and centromere-adjacent mitotic gene conversion were performed. Disomic haploid cells heteroallelic at two loci on the left arm of chromosome III were exposed to ethyl methanesulfonate (EMS) under nonlethal conditions; EMS-induced prototrophic gene convertants were selected and tested for coincident chromosome loss. The principal results are: (1) The frequency of chromosome loss among EMS-induced gene convertants selected to arise near the centromere is markedly enhanced over basal levels and remains constant, independent of EMS exposure. There is little such enhancement among EMS-induced convertants selected to arise far from the centromere. (2) Chromosome loss is almost completely associated with induced conversion of the centromere-proximal allele at the centromere-adjacent heteroallelic locus. This result is identical to (and confirms) results found previously for spontaneous loss-associated conversion. (3) The conversion polarity at the centromere-adjacent locus among unselected (nonloss-associated) induced or spontaneous mitotic convertants is identical to that among meiotic convertants and markedly favors the contromere-distal allele. These findings are wholly consistent with, and strengthen, the hypothesis that structural involvement of centromeric regions in nearby recombinational events may interfere with proper segregational function and lead to mitotic chromosome loss.     

Analysis of polymorphism at nine nuclear genome DNA loci in Maris

Population genetic survey of the indigenous populations of the Marii El Republic, represented by the two major ethnographic groups of Maris, Meadow (five samples from Morkinsk, Orshansk, Sernursk, Sovetsk, and Zvenigovsk districts) and Mountain (one sample from Gornomariisk district) Maris, was carried out. All Mari groups were examined at nine polymorphic DNA loci of nuclear genome, VNTR(PAH) (N=422), STR(PAH) (N=152), VNTR(ApoB) (N=294), VNTR(DAT1) (N=363), VNTR(eNOS) (N=373), ACE (N=412), IVS6aGATT (N=513), D7S23(KM.19) (N=494), and D7S8 (N=366). Allele and genotype frequency distribution patterns were obtained for individual samples and ethnographic groups, as well as for the ethnic group overall. In each of six Mari samples examined, the deficit of heterozygotes was observed, i.e., the mean observed heterozygosity was lower than the expected one. The indices of mean heterozygosity, $\bar H_S = 0.455$ , and interpopulation differentiation, $\bar F_{ST} = 0.0024$ , for the Mari gene pool were obtained using a set of DNA markers analyzed. The highest level of interpopulation differentiation is characteristic of ACE loci (F _ST=0.0104) and D7S23(KM.19/PstI) (F _ST=0.0123). Analysis of the genetic distances and interpopulation differentiation (F _ST) showed that the main part of genetic diversity in Maris was determined by the differentiation between the populations of Meadow Maris. The contribution of the differences between the ethnographic groups of Mountain and Meadow Maris to the ethnic gene pool was small. It is suggested that the main role in the formation of the Mari gene pool is played by the geographic factor.     

Purification and Characterization of Carbaryl Hydrolase from Blastobacter sp. Strain M501

A bacterium capable of hydrolyzing carbaryl (1-naphthyl-N-methylcarbamate) was isolated from a soil enrichment. This bacterium was characterized taxonomically as a Blastobacter sp. and designated strain M501. A carbaryl hydrolase present in this strain was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, gel filtration, and hydroxylapatite chromatographies. The native enzyme had a molecular mass of 166,000 Da and was composed of two subunits with molecular masses of 84,000 Da. The optimum pH and temperature of the enzyme activity were 9.0 and 45°C, respectively. The enzyme was not stable at temperatures above 40°C. The purified enzyme hydrolyzed seven N-methylcarbamate insecticides and also exhibited activity against 1-naphthyl acetate and 4-nitrophenyl acetate.     

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens)

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD⁴⁺ T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d _S) and non-synonymous (d _N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d _N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.     

Mineralization of Carbofuran by a Soil Bacterium

A bacterium, tentatively identified as an Arthrobacter sp., was isolated from flooded soil that was incubated at 35°C and repeatedly treated with carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate). This bacterium exhibited an exceptional capacity to completely mineralize the ring-labeled ¹⁴C in carbofuran to ¹⁴CO₂ within 72 to 120 h in a mineral salts medium as a sole source of carbon and nitrogen under aerobic conditions. Mineralization was more rapid at 35°C than at 20°C. No degradation of carbofuran occurred even after prolonged incubation under anaerobic conditions. The predicted metabolites of carbofuran, 7-phenol (2,3-dihydro-2,2-dimethyl-7-benzofuranol) and 3-hydroxycarbofuran, were also metabolized rapidly. 7-Phenol, although formed during carbofuran degradation, never accumulated in large amounts, evidently because of its further metabolism through ring cleavage. The bacterium readily hydrolyzed carbaryl (1-naphthyl N-methylcarbamate), but its hydrolysis product, 1-naphthol, resisted further degradation by this bacterium.     

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6^cs, ca^nd, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6^cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca^nd, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.     

Structure-activity dependence in some novel ring-substituted 3,3-dimethyl-1-phenyltriazenes. Genetic effects in Drosophila melanogaster and in Saccharomyces cerevisiae by a direct and a host-mediated assay

The structure-activity dependence of ten ring-substituted 3,3-dimethyl-1-phenyltriazenes (DMPT), 3,3-dimethyl-1-(3-pyridyl)-triazene (3-PyDMT) and of 3,3-dimethyl-1-(3-pyridyl-N-oxide)-triazene (3-PyODMT) was investigated by the induction of recessive lethal mutations in Drosophila melanogaster and of mitotic gene conversions in Saccharomyces cerevisiae using both direct and host-mediated assays. Significant differences in genetic effectiveness were detected not only between structurally related compounds but also between the responses of each test system to the same mutagen. Triazenes which are easily cleaved at physological conditions showed the highest genetic activity in the direct yeast test whereas stable triazenes, especially those with ortho and para positions blocked by a halogen, were most active in Drosophila. We have concluded that (1) the released arenediazonium cation is most probably responsible for the convertogenic activity in yeast; (2) metabolites, arising from hydroxylation of the methyl group, are essential for the mutagenic activity in Drosophila. A possible molecular basis which could account for the diversity in genetic effectiveness is discussed in terms of reaction mechanisms which can be predicted from the structural features of the tested triazenes.     

Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.     

Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation

Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in Δsir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.     

元江鲤种群遗传多样性

选择12对微卫星标记检测了于2011年采集自元江(红河上游中国江段)5个样点192尾鲤的群体遗传多样性.共检测到201个等位基因,每个位点等位基因2-27个.各群体各位点平均等位基因(NA)12.25-14.67个,平均有效等位基因(NE)8.28-9.73个,平均观察杂合度(Ho)o.7765-0.8037,平均期望杂合度(HE)0.7761-0.8080,平均多态信息含量(PIC)0.7534-0.7843.元江鲤种群192个个体各位点NA、NE、Ho、HE、PIC分别为16.50、11.26、0.7927、0.8049、0.7966,种群遗传多样性水平高.元江鲤群体之间遗传分化小,可作为一个种群管理单元进行管理.增殖放流要防止遗传多样性丧失.     

Simple and efficient synthesis of chiral amino alcohols with an amino acid-based skeleton

Sodium bis(2-methoxyethoxy)aluminum hydride, NaAlH₂(OCH₂CH₂OCH₃)₂, commercially known as Vitride® or Red-Al®, enables rapid synthesis of pure optically active N-protected amino alcohols and peptide alcohols in very high yields. The method is very simple and attractive, as it does not require an additional step of N-protected amino acid derivatization and proceeds without the loss of enantiomeric homogeneity.     

Genetic Diversity of Some Pear Cultivars and Genotypes Using Simple Sequence Repeat (SSR) Markers

The genetic diversity and relationships among 47 pear cultivars and genotypes (Pyrus spp.), including 4 Japanese pears (Pyrus pyrifolia), 40 European pears (Pyrus communis), 1 Chinese pear (Pyrus bretschneideri) as well as 2 wild relatives (Pyrus salicifolia and Pyrus mazandaranica) were studied using 28 microsatellite primer pairs. A total of 174 alleles were produced at the 28 SSR loci with their sizes ranging from 81 to 290?bp. The number of observed alleles for each locus ranged from 3 (TsuENH014 and TsuENH046) to 12 (NB103a), with an average of 6.21 alleles per locus. In some SSR loci, more than two alleles were amplified in some cultivars and genotypes, suggesting that duplication has occurred in those accessions. This information suggests that at least two genomic regions exist for these loci in the pear genome. The observed heterozygosity (H _o) values of amplified loci ranged from 0.17 (TsuENH006) to 0.97 (NB103a). Shannon's information index (I) value was observed to be highest (2.14) in the NB103a locus, while the TsuENH006 locus had the lowest value with an average of 1.37 among SSR loci. The Dice genetic similarity coefficient ranged from 0.29 (??Nijisseiki?? and P. mazandaranica) to 0.91 (??Chojuro?? and ??Nijisseiki??) among samples. UPGMA cluster analysis showed two major groups corresponding to the Japanese and European pears.     

Kinetic analyses of transmitter release in neuromuscular transmission

A majority of transmitter quanta in the nerve terminal is in a large pool of store (S), which can be utilized for release only after transformation into activated quanta (n) via two intermediate states called available quanta (A) and releasable quanta (N). Mobilization is a collective term applicable for aggregates of S → A, A → N and N → n conversions. In the present article, various electrophysiological procedures for kinetic analyses of the transmitter release in neuromuscular transmission were discussed to elucidate this not well understood process of mobilization. Especially, frequency augmentation, tetanic rundown and depression, and recovery from depression experiments were proposed to be very useful tools in identifying the drug effect on the mobilization process. since d-tubocurarine, 2-(4-phenylpiperidino) cyclohexanol (AH5183) and Ba ions satisfied the above three criteria of drug action on mobilization, these were concluded to affect the mobilization of transmitter in neuromuscular transmission.     

Fission yeast sta mutations that stabilize an unstable minichromosome are novel cdc2-interacting suppressors and are involved in regulation of spindle dynamics

Cytological observations have shown that the presence of unstable minichromosomes can delay progression through the early stages of mitosis in fission yeast (Schizosaccharomyces pombe), suggesting that such minichromosomes may provide a useful tool for examining the system that regulates the coordinated segregation of chromosomes. One such unstable minichromosome is a large circular minichromosome. We previously showed that the mitotic instability of this minichromosome is probably due to the frequent occurrence of catenated forms of DNA after replication. To identify genes involved in the regulation of chromosome behavior in mitosis, we isolated mutants which stabilized this minichromosome. Three loci (stal, sta2, and sta3) were identified. Two of them were found to be suppressors of temperature-sensitive mutations in cdc2, which encodes the catalytic subunit of muturation promoting factor (MPF). They show no linkage to, and are thus different from, sucl, and cdc13, previously identified as genes that interact with cdc2. The other mutation mapped to a gene previously identified as being required for the correct formation of the mitotic spindle. Data provided in this study suggest that the sta genes are involved in the regulation of spindle dynamics to ensure proper chromosome segregation during mitosis.     

Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the rRNA (45s), the Major Chlorophyll a/b Binding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes

DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm of chromosome 2 and corresponds to the Nucleolar Organizer Region. The size of the 45s repeating unit is estimated to be approximately 9 kb in Lycopersicon esculentum and 11 kb in Lycopersicon pennellii. Five loci were found to contain CAB sequences. Two of the loci, Cab-1 (chromosome 2) and Cab-3 (chromosome 8), together accounted for more than 80% of the hybridization signal. These loci contain more than one CAB structural gene. The other three loci, Cab-2 (chromosome 8), Cab-4 (chromosome 7) and Cab-5 (chromosome 12), each account for <10% of the total signal and may contain only a single copy of the CAB structural sequence. Three loci were found to contain RBCS sequences. Rbcs-2 (chromosome 3) and Rbcs-3 (chromosome 2) were responsible for >80% of the signal, with the remainder being associated with Rbcs-1 (chromosome 2). Rbcs-2 and Rbcs-3 may contain more than one copy of the gene.     

Correlation of gene frequencies between neutral linked genes in finite populations

The mean squared correlation (r²) of gene frequencies between two selectively neutral loci in segregating populations is obtained using an approximation based on expanding the formula for r² in terms up to eighth moments. The moments are evaluated for all populations, whether segregating or not, but when combined with the probability of segregation, quite accurate results are obtained. Formulae for the moments and an approximation for the probability of segregation are given for populations of large size (N) and small recombination fraction (c). The method is extended to evaluate the variance of r². The coefficient of variation of r² exceeds unity for a wide range of Nc values.     

Isolation of Aneuploid-Generating Mutants of ASPERGILLUS NIDULANS, One of Which Is Defective in Interphase of the Cell Cycle

A method is described for isolating mutants potentially defective in loci involved in mitotic chromosome segregation. Conditional lethal, heat-sensitive (42°) mutants were assayed at a subrestrictive temperature of 37° for an inflated production of colonies displaying phenotypes and behavior patterns of whole chromosome aneuploids. Of 14 mutants, three showed specificity for one disomic phenotype, whereas 11 generated colonies mosaic for different aneuploid phenotypes. This latter group is designated hfa ( high frequency of aneuploid). For ten of the 11 mutants temperature sensitivity and aneuploid production cosegregated, indicating a single mutation in each. These mutations were recessive and nonallelic. Analysis was concentrated on the hfaB3 mutation which is mapped to chromosome VI tightly linked to the methB and tsB loci. The disruptive influence of hfaB3 on mitosis at 37° was shown by (1) ploidy and whole chromosome-type segregation of markers in the breakdown sectors of phenotypically aneuploid colonies obtained from multiply marked homozygous hfaB3 disploids; (2) a high frequency of haploid and nondisjunctional diploid segregants among spontaneous yellow-spored parasexual recombinants taken from green-spored homozygous hfaB3 diploids. The mutation had no effect on meiotic chromosome segregation at 37°. The single interphase nucleus in germlings at 42°, coupled with changes in the mitotic index in temperature exchange experiments, showed hfaB3 to arrest the cell cycle in interphase at restrictive temperature. A conclusion drawn is that the hfaB gene product is required both for entry into mitosis and for normal chromosome segregation in dividing nuclei.