利用制霉菌素溶缩处理高频率地检出黑曲霉营养缺陷型突变株

以亚硝酸诱变处理黑曲霉Ｎ３４３和黑英霉ＵＶ－１１的分子孢子,然后将它们培养在它们培养基平板上至第二代分生孢子生长,收集这些孢子以制霉菌素浓缩处理,结果以３．８－０．３％的高频率和到１９株稳定的营养缺陷型突变株：从黑曲霉Ｎ３４３获得３株维生素类的缺陷型,它们分别为双缺或参缺;从黑曲霉ＵＶ－１１获得１６株特殊遥脯氨酸营养缺陷型,进一步分析表明这是由于从谷氨酸合成脯所酸或精氨酸的代谢阻断所致。     

利用制霉菌素浓缩处理高频率地检出黑曲霉营养缺陷型突变株

以亚硝酸诱变处理黑曲霉Ｎ３４３和黑曲霉ＵＶ－１１的分生孢子,然后将它们培养在完全培养基平板上至第二代分生孢子长出,收集这些孢子以制霉菌素浓缩处理,结果以３．８～０．３％的高频率得到１９株稳定的营养缺陷型突变株：从黑曲霉Ｎ３４３获得３株维生素类的缺陷型,它们分别为双缺或叁缺;从黑曲霉ＵＶ－１１获得１６株特殊的脯氨酸营养缺陷型,进一步分析表明这是由于从谷氨酸合成脯氨酸或精氨酸的代谢阻断所致。所有以上突变株形态与各自原养型相似,但在产酶活力方面均为负突变     

米曲霉原生质体融合及杂合二倍体的形成

采用混合酶液处理纤维素酶高产苗株３０４２_Ｎ－２（天冬酰胺缺陷型“Ａｓｎ^－”）及蛋白酶高产菌株３０４２_Ｎ－１９（蛋氨酸缺陷型“Ｍｅｔ^－”）的营养菌丝,获得原生质体,以ＰＥＧ为助融剂进行融合处理,成功地获得了米曲霉（Ａｓｐｅｒｇｉｌｌｕｓｏｒｙｚａｅ）原生质体的营养互补融合。将异核体菌落的菌丝转接到合有０．１％樟脑的新鲜ＭＭ上,２５℃诱导培养７─１５天,挑取绿色角变菌落的孢子,将其转接到ＭＭ上能迅速生长,经孢     

放线菌种间原生质体电融合育种

以龟裂链霉菌(S．Rimosus,Otc^r,Sm ^s)和灰色链霉菌(S．Griseus,Sm^r,Otc^s)为原始出发菌株,经NTG诱变处理,获得前者(Asp^-)和后者(cys^-)营养缺陷型突变株,制成10⁹个／m1等量原生质体混合液,在岛津细胞融合装置SSH—C¹¹融合槽(电极间距为0．5mm)内,以频率lMHz、强度800V／cm的高频交流电场作电介质,电泳15s形成原生质体珠串,旋即施加电场强度6 kV／cm、短时程20μs的直流高压方形电脉冲触发原生质体融合,在R₃再生培养基上选取34971个菌落,移植于双抗基本培养基,检出四个双抗原养型融合子。它们具有亲株的优良生物学特性,生长速度较快,产素能力大,抗菌活性高。生物显影结果表明,它还产生两个亲株所不具有的抗菌活性物质。     

黑曲霉UB4和NB3的原生质体制备及其再生条件的研究

以两株黑曲霉营养缺陷型突变株ＵＢ４和ＮＢ３为材料,对于其原生质体制备及其原生质体再生条件作了详细的研究,从中找到了合适的破细胞壁方法和培养条件,为尔后的黑曲霉原生质体的利用如融合或诱变育种等方面的研究奠定了基础。     

基因工程菌发酵合成L－苏氨酸－N15

采用含有稳定同位素^１５Ｎ－硫酸铵为主要氮源的专用发酵培养基配方和提取精制条件,在国内、外首次采用基因工程菌ＡＡ_７（ｐ^ＴＨ２）（ＡＨＶ^ｒＡＥＣ^ｒ,Ｔｈ^ｒ－Ｎ^－,Ｈｏｍ^ｒ,Ａｐ^ｒ）直接发酵方法研制Ｌ－苏氨酸－Ｎ^１５高丰度精制产品。每ｍｏｌ^１５Ｎ－硫酸铵实际得到０．６３８ｍ     

脱卤脱亚硫酸菌脱氯呼吸链的遗传分析*

以携有结合转座子Ｔｎ９１６的Ｅｎｔｅｒｏｃｏｃｃｕｓ　ｆａｅｃａｌｉｓ　ＪＨ２－２为供体,脱卤脱亚硫酸菌ＨＳＳ１（Ｄｅｓｕｌｆｉｔｏｂａｃｔｅｒｉｕｍ　ｄｅｈａｌｏｇｅｎａｎｓ,Ｓｍ抗性突变株）为受体,在厌氧条件下,通过滤膜杂交、结合转移,将Ｔｈ９１６转移并插入到受体菌的染色体上,其转移频率为：１．１×１０^－７～３×１０^－８。　在丙酮酸／乳酸－３－氯－４－羟基苯氧乙酸、Ｔｃ、Ｓｍ培养基上,筛选脱氯呼吸的缺陷型突变株,并用反向ＰＣＲ（Ｉ     

转座子Tn916接合转移诱变棒杆菌

采用滤膜接合法,将大肠杆菌中带有Ｔ_ｃ抗性的转座子Ｔｎ９１６接合转移到北京棒杆菌ＧＭ９３中其进行诱变。接合频率在１０^－７－１０^－８之间,在没有选择压力的条件下传代培养,接合子是比较稳定的,仍保持Ｔ_ｃ抗性。Ｔｎ９１６的接合转座作用可以使ＧＭ９３产生营养缺陷型突变,其中氨基酸营养缺陷型占８４％,并得到与Ｌ－Ｉｌｅ产生途径有关的六种氨基酸（Ｔｈｒ,Ｌｙｓ,Ｌｅｕ,Ｍｅｔ,Ｖａｌ,Ｉｌｅ）的缺陷株。     

耐热酵母与酿酒酵母原生质体融合的研究

酿酒酵母(saccharomyces cerevisiae)396是利用甘蔗糖蜜生产酒精的生产菌株,假丝酵母(Candida sp·)C₆是我们从云南温泉底泥中筛选到的一株能在45℃生长良好的耐热酵母,我们应用原生质体融合技术进行了两菌株属间融合的研究。通过亚硝基胍(NTG)诱变得到的营养缺陷型菌株396(arg^－)和C6(lys^-),其融合频率为O．91×10^-5。从检出的融合子中挑选6株进行考核、其细胞体积平均为亲株的1．3倍,DNA含量平均为亲株的1．6倍,培养特征、形态、大小和生理生化特征表现不一,特别是糖类的同化试验。除F₂、F₁₄外,其他4株可以排除异核体形成的可能性。比较了在28℃,40℃和45℃培养条件下出发亲株396、C₆、直接亲株396(arg^-)C₆(lys^-)和融合子F₁、F₇、F₁₂,F₁₃的生长曲线、基质利用率和乙醇产率等,得到一株在40℃培养条件下糖的利用率为94．3％、乙醇产量为59．7g／L的属间融合株F₁₃。     

应用原生质体融合技术定向改造林可霉素产生菌的探索

金色链霉菌streptomyces aureofaciens(LM^r,CTC^r,产金霉素)的原生质体经u。V．照射40min灭活后,与林可链霉菌林可变种Streptomyces Iinclnensis Var lincilne—nsis(LM^r,CTC^I,产林可霉素),在42％PEG(Mw6000)诱导下进行种间原生质体融合,在含金霉素50μg／Mi的再生平板上直接选择融合子,融台率达9．05×10^-5。从大量融合体中筛选到4株产生抗菌物质不同于亲株并较稳定的菌株。对其中一株所产的抗生索作了初步鉴别,推测其结构与林可霉素相近。另一株的薄层层析R^VB_f值与氯林可霉素相近,尽管此等产物还有待于进一步鉴别,但这一育种途径值得探讨。     

Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.     

Effectiveness of Sesbania rostrata and Phaseolus calcaratus as green manure for upland rice grown in acidic soil

Two field experiments on green manuring were conducted under upland acidic soil (pH = 4.35) conditions with the following objectives: (1) to determine the influence of inoculation site, P fertilization, and liming on the biomass production, N content, N accumulation, and N availability of S. rostrata grown in an acidic soil, (2) to compare the effectiveness of S. rostrata, P. calcaratus and urea as N sources for upland rice as affected by liming and N source-sowing time combination, and (3) to assess the effect of liming and N source-sowing time combination on % Ndff (N derived from the fertilizer), % Ndfs (N derived from soil), % FNU (fertilizer N utilization), and FNY or fertilizer N yield (kg N ha^–1) of upland rice grown in acidic soil. At 2 weeks after incorporating S. rostrata (95 days after lime application), liming significantly increased N availability by more than 2-fold suggesting that the decomposition of S. rostrata by soil microflora was stimulated by lime. Liming, phosphorus application, and inoculation site improved significantly the dry biomass production, N content and N accumulation of S. rostrata; thus, enhancing its green manuring potential. Regardless of liming, S. rostrata whether applied at 0 week or 2 weeks before sowing was superior to urea in improving grain and straw yields. P. calcaratus when applied at 2 weeks before sowing also produced higher grain yield than urea. Immediate sowing of upland rice after green manure incorporation did not affect negatively the growth and development of upland rice; hence, farmers could save at least 2 weeks in their cropping calendar. N source-sowing time combination had a highly significant influence on % Ndff, % Ndfs, % FNU, N uptake, and fertilizer N yield of upland rice. However, only N uptake was influenced significantly by liming. The rice plant obtained significantly higher % Ndfs from the soils treated with green manure than those treated with urea regardless of liming. The % FNU and % Ndff from the green manures were 11-37% and 9-25%, respectively. These values are much lower than those obtained under continuously flooded soil conditions possibly because of the differences in the organic matter decomposer populations and N loss mechanisms between sloping upland conditions and continuously flooded conditions.     

高等担子菌双重营养缺陷突变菌株的分离

带有单一营养缺陷的凤尾菇和裂褶菌的单核体菌株经亲和性交配,各自交配产生后代,从中分离出遗传特性稳定,生理特征表型正常的双重缺陷营养害变型菌株,为原生质体融合育种研究提供了可靠的亲本菌株。     

A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M11 (Microsporida: Nosematidae), into an Insect Cell Culture

ABSTRACT. Spores of Nosema sp. NIS M11, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL-41 medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldini's solution. By using the salt solutions as inoculation media, KOH-primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF21AEII cell line. The initial infection levels were consistently higher than that obtained by using IPL-41 medium. Among the salt solutions, Rinaldini's solution, containing KCl in place of NaCl, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M11.     

Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]

A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.     

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.     

Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.     

Dechlorination of chlorophenols found in pulp bleach plant E-1 effluents by advanced oxidation processes

Studies were conducted on the response of 2,4,6-trichlorophenol (1), 2,3,4,5-tetrachloro-phenol (2) and 4,5-dichloroguaiacol (3) toward advanced oxidation processes, such as UV-, O2/UV-, H2O2/UV-, O3/UV- and O3-H2O2/UV-photolyses with irradiation of 254 nm photons. The compounds 1-3 are among the chlorophenols found in the Kraft-pulp bleach plant E-1 effluents. The studies were extended to treatment of these compounds with ozonation and O3-H2O2 oxidation systems in alkaline aqueous solution. Except for the O2/UV-photolysis of 1 and H2O2/UV-photolysis of 2, the dechlorination of 1-3 by O2/UV- and H2O2/UV-potolyses were less effective than the corresponding N2UV-potolysis of 1-3. Guaiacol-type chlorophenols were more readily able to undergo dechlorination than non-guaiacol type chlorophenols by N2/UV-, O2/UV- and H2O2/UV-potolyses. In addition, the efficiency for the dechlorination of 1-3 by N2/UV-, O2/UV- and H2O2/UV-potolyses appeared to be dependent upon the inductive and resonance effects of substituents as well as number and position of chlorine substituent in the aromatic ring of the compounds. The dechlorination of 2 by treatment with O3 alone is slightly more effective than the corresponding the O3/UV-photlysis, whereas the dechlorination of 2 by treatment with the combination of O3 and H2O2 was slightly less effective than the corresponding O3-H2O2/UV-photolysis. In contrast, the dechlorination of 3 on treatment with O3 alone was slightly less effective than the corresponding the O3/UV-photolysis, whereas the dechlorination of 3 on treatment with the combination of O3 and H2O2 was slightly more effective than the corresponding the O3-H2O2/UV-photolysis. In the dechlorination of 2 and 3, chemical species derived from ozone and hydrogen peroxide in alkaline solution were dominant reactions in the O3/UV- and O3-H2O2/UV-photolysis systems as in the O3 and O3-H2O2 oxidation systems. Possible dechlorination mechanisms involved were discussed on the basis of kinetic data.     

Protoplast fusion permits high-frequency transfer of a Streptomyces determinant which mediates actinomycin synthesis.

Prototrophic recombinants and heterocaryotic colonies developed at high frequency when protoplasts of nutritionally complementary actinomycin-producing and nonproducing strains of Streptomyces antibioticus were fused in the presence of polyethylene glycol and plated on minimal regeneration medium. Of the spores obtained from aerial hyphae of a single heterocaryotic colony, 99% carried the act+ character regardless of whether the nutritional markers of the spore were derived from the act+ or the act parent. Similarly, a high-frequency transfer (68% in S. antibioticus, 48% in Streptomyces parvulus) of act+ determinant(s) to act was achieved by protoplast fusion. Protoplasts of a doubly auxotrophic act strain of S. parvulus were efficiently transformed in the presence of polyethylene glycol with respect to the auxotrophic markers by DNA of an act+ auxotrophic strain with complementary nutritional requirements. The transformation frequency of the nutritional (chromosomal) markers was 17%. In contrast, the transformation frequency for actinomycin synthesis was less than 1%.     

利用营养缺陷诱变对金顶侧耳进行基因连锁分析研究

利用金顶侧耳的营养缺陷型菌株配制杂交菌株,通过营养缺陷型标记对其后代进行连锁分析,确定不亲和性因子和营养缺陷标记所在的连锁群及其排列顺序。截止目前的试验数据表明,金顶侧耳至少由6条染色体(连锁群)组成。其中A因子存在的第1连锁群上分布着ade2、pab1、ade5、met2、met7营养缺陷型基因位点;B(Bα、Bβ)因子存在的第2连锁群上分布着cho1、ade1营养缺陷型基因位点;第3连锁群上分布着met9、arg1、his1、ade7、his3、met1营养缺陷型基因位点;第4连锁群分布着nic1、nic2、ade4、pdx2营养缺陷型基因位点;第5连锁群分布着pan1、ino1营养缺陷型基因位点;第6连锁群分布着ile1营养缺陷型基因位点。金顶侧耳与其他担子菌的遗传图谱相同,第1连锁群A因子附近均分布着Ade及Pab营养缺陷型基因位点。