Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.     

S－美芬妥英羟化代谢多态性的分子机制研究进展

人群中存在着Ｓ－美芬妥英羟化代谢多态性。从人肝微粒体已分离出Ｓ－美芬妥英羟化酶。在基因克隆研究中已分离出与该羟化酶活性有关的Ｐ４５０ｃＤＮＡ,属Ｐ４５０２Ｃ１９。最近报道,人肝中Ｐ４５０２Ｃ１９的含量和催活Ｓ－美芬妥英羟化活性密切相关,其弱代谢者主要由于ＣＹＰ２Ｃ１９的外显子５单碱基对（Ｇ→Ａ）的变异,使核苷酸序列错位而产生无功能的Ｐ４５０蛋白的结果。     

Use of Local Raw Materials for the Production of Bacillus sphaericus Insecticide in Ghana

Locally available raw materials were used as fermentation media for the preparation of an effective bacterial insecticide of Bacillus sphaericus in Ghana. The choice of materials for media production was based on their availability, their cost and how well they supported growth and sporulation of the bacterium. The materials used were anchovy (Engraulis encrasicolus), spent grain from breweries, bambara beans (Vigna subterranea), sprout maize (Zea mays) and B. sphaericus strain IAB 881. The larvicidal activities of each final whole culture of the insecticides prepared were evaluated against third and fourth instar larvae of the mosquito species Culex quinquefasciatus. The levels of larvicidal activity exhibited by preparations using the raw materials were similar to those where the bacterium was cultured in a synthetic medium, with median lethal concentrations ranging from 0.30 10- 5 to 0.68 10-6. Cell counts were in the range 11 108 - 36 108 colony-forming units ml-1, and spore counts were between 29 107 and 61 107 ml-1.     

Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica

Eight polymorphic microsatellite markers from the swallow were isolated and characterized. Extraordinary variability was revealed at the HrU6 locus with 45 different alleles scored among 46 unrelated individuals. The probability that the same genotype combination would occur in two random and unrelated individuals at six selected loci was as low as 1.3 × 10^-8 and the combined exclusion probability was 0.9996. Stable Mendelian inheritance was observed in about 1000 meioses. No significant linkage was revealed and for almost all combinations of marker-pairs, linkage closer than 5 cM could be excluded. At two loci, null (nonamplifying) alleles were encountered. Thirteen (30%) extra-pair offspring were identified in 5 (56%) broods when applying the marker set on a nearly complete swallow colony. We were able to identify a single male from the other families in the colony as the most likely father for nine of the 13 extra-pair offspring.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

乙型脑炎病毒NS2A-C60A位点突变对其生物学特性影响研究*

目的:旨在探索Ⅰ型日本乙型脑炎病毒传代致弱后基因组突变NS2A-C60A对乙脑病毒生物学特性的影响。方法:首先通过对传代致弱及原始乙脑毒株基因组序列进行测序比对、结构预测分析并利用Western blotting(WB)确定了目标研究位点NS2A-C60A;然后使用反向遗传定点突变技术构建拯救了包含NS2A-C60A单点突变的病毒株;最后利用噬斑形态观察、生长曲线、双萤光素酶分析,WB以及炎性因子检测和动物实验研究了该单点突变对于乙脑病毒生物学特性的影响。结果:首次研究发现Ⅰ型乙脑病毒传代致弱会导致NS1'蛋白表达的显著下降以及可能的相关位点NS2A-C60A,并成功拯救获得了NS2A-C60A单点突变毒株rJEV-C60A,研究发现NS2A-C60A突变对乙脑病毒的生长特性及噬斑形成没有显著影响,但是能够显著降低乙脑病毒NS1'蛋白的表达,并且该位点突变能够轻微阻碍乙脑病毒对细胞炎性因子表达的抑制,动物实验结果显示NS2A-C60A点突变病毒与原毒株具有相似的神经毒力,说明该位点突变不是影响乙脑病毒毒力致弱的关键位点。结论:新发现的NS2A-C60A位点突变能够显著减少乙脑病毒NS1'蛋白的表达,但是对其增殖、诱导炎症及神经毒力等生物学特性没有显著影响。     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

Isolation and characterization of pock-forming plasmids for Streptomyces griseus from soil actinomycetes

Thirty independent actinomycetes strains carrying plasmids were isolated from soil. These plasmids were purified as cccDNA by CsCl-EtBr equilibrium density-gradient centrifugation. Plasmids that induce "pocks", namely formation of circular zones of sporulation-inhibition, were selected by protoplast transformation of streptomycin-producing strain, Streptomyces griseus ATCC10137. Six pock-forming plasmids, pOA7, pOA11, pOA15, pOA23, pOA29 and pOA30, were obtained, and their cleavage maps, transformation frequencies, and copy numbers, as well as their stability, are described.