高产融合子Q4413的形态学与生理生化特征的研究

本文通过对枯草芽孢杆菌BR151衍生株与北京棒杆菌1134衍生株的赖氨酸高产融合子Q4413株的形态学、生理生化特性等方面的研究,揭示了融合子与双亲株在这些方面的差异,为Q4413株确系双亲株的重组子或新的融合子增加了佐证.     

原生质体融合选育赖氨酸高产菌种的研究

北京棒杆菌1134衍生株与枯草芽孢杆菌151衍生株的原生质体融合得到了一株可以甜菜糖蜜为原料,在摇床与小罐发酵均能稳定产赖氨酸6 50％以上的高产菌株——Q4413。Q441 3菌落形态类似于11 34株,而菌体形态区别于双亲,呈较短的杆状,无鞭毛,但有较稀疏的纤毛。生理生化研究表明：Q4413耐盐性、蔗糖发酵,石蕊牛奶试验产碱状况酷似于151株,而与1134株略有差异;运动性、糊精、柠檬酸盐利用,MR试验、酪素水解、明胶水解、石蕊牛奶还原,又酷似于1134株而区别于151株;04413的最适生长温度、pH值居双亲之间;Qq4413为原养型加三重抗性[Rifr,AECr Nalr];其细胞DNA含量与GC比测定结果均居双亲之中,而且生物量测定结果也均与双亲有一定的差异。     

细菌科间原生质体融合的研究

本文将赖氨酸产生菌——北京棒杆菌1134株进行标记,同时将枯草芽孢杆菌BR151菌株进一步标记后,进行科间原生质体融合的探讨,揭示了科间原生质体融合的可能性。经适宜的条件培养和预处理,再在适宜的条件下,酶解制备亲本株的原生质体,用PEG(MW6000)为聚合剂,经低温短时间处理,将1134衍生株与151衍生株的原生质体进行融合;并在改良的DM_3平皿上加入适量的细胞壁碎片(作引物)与适量的人血清白蛋白制剂,使其再生,结果棒杆菌(在酶解后2.5h.)与枯草芽孢杆菌(酶解后20min)的制备率均已达到99.98％以上,再生率分别达21.3％与90.8％以上。经适温培养48h后,从融合平皿上随机挑出若干个菌落。点种于CM母平皿上,再分别影印于10套选择培养基平皿上,进行鉴定,其融合率达到了3.9×10~(-4)。从菌落形态和菌体形态上可大体分为近1134类型,近151类型和中间类型三种。经连续传代五代后,从融合子中筛选山了几株在摇床上以甜菜糖蜜为原料。产酸水平捉高近一倍的稳定高产融合子。其中Q4413株的产酸率平均达6.56％,10立升小罐发酵产酸率6.52％,糖酸转化率为40.25％,经进一步验证,该菌株的产酸能力和遗传性均是稳定的,为远缘工业微生物育种开辟了新的可行途径。     

利用原生质体融合技术选育防治植物病虫害的基因重组菌株

本试验选用抗菌蛋白产生菌枯草芽孢杆菌(B.subtilis) TG26和晶体蛋白产生菌苏云金芽孢杆菌(B.thuringiesis subsp,pacifeus) AS1.904的营养缺陷型衍生株,在聚乙二醇的诱导下进行原生质体种间融合,获得了表现双亲遗传性状的种间融合菌株。融合率为7.52×10~6,融合子经传代10次,稳定率为19.5%。融合菌株的菌落和细胞形态与亲本株明显不同。通过SDS-聚丙烯酰胺凝胶电泳检测,融合菌株表达了亲本的抗菌蛋白和毒素蛋白。抑菌杀虫试验表明,融合重组菌株具有抑制多种植物病原菌和毒杀鳞翅目幼虫的能力。     

高效嗜热聚磷菌的构建

以一株能聚磷但不耐热的芽孢杆菌和一株嗜热但不聚磷的嗜热菌为亲本,PEG诱导原生质体融合,筛选出2株既耐高温又能聚磷的融合菌株。对2株融合子进行传代培养,同时对未传代培养的融合子和传代培养后的融合子进行聚磷及耐热性测定,2株菌株的聚磷能力均比亲株高,其对无机磷的聚积能力分别为27.8%和5.8%,对有机磷的聚积能力分别为39.2%和56.1%,,耐热温度达55℃。该稳定的融合子具有嗜热和聚磷的双重遗传标记,可以作为研究基因重组的材料及高温生物除磷的生产菌株,对研究聚磷菌的聚磷机理和嗜热菌耐热机理,解决夏季污水处理曝气池发泡过多现象提供一定的依据。     

原生质体融合技术构建棕榈油酸高产酵母菌株

采用原生质体融合技术进行产棕榈油酸酵母Saccharomy cescerevisiaeNo.12.926和产脂酵母RhodotorulaNo.12.908的融合研究,获得了棕榈油酸高产酵母工程菌株。实验结果表明,原生质体形成的最佳条件为:对数期酵母No.12.926和No.12.908用2%蜗牛酶于30℃分别酶解1.5和2h。在最佳条件下,酵母No.12.926和No.12.908原生质体形成率分别为94%和80%,再生率分别为75%和60%。原生质体融合由聚乙二醇诱导。将得到的融合子进行多次传代培养优选,获得了遗传性状稳定的融合菌株。融合子的生物量为亲株的两倍多,其细胞形态和菌落颜色与亲株有差别。产脂和产棕榈油酸分析表明,融合子的产脂量为菌体干重的48.53%,其中棕榈油酸占油脂总量的47.29%,为菌体干重的22.95%。     

通过原生质体融合产生的苏云金杆菌的新菌株

用苏云金杆菌以色列变种Bacillus thuuringiensis subsp.israelensis和苏云金杆菌库斯塔基变种Bacillus thuringuensis subsp.kurstaki HD-1进行了PEG诱导的原生质体融合实验,探讨了影响原生质体形的几个条件。用抗性标记对融合子进行了选择,在198株融合子中,有17株稳定传代10次以上。它们具有两个亲本菌株所具有的一些特性。在融合子中,BTI比HD-1稳定。用SDS-聚丙烯酰胺凝胶电泳方法研究比较了稳定融合子的晶体毒素成分,观察到了代表HD—l毒素蛋白和BTI毒素蛋白的带。杀虫试验结果表明,融合形成的杂种菌株具有毒杀文字孑孓和鳞翅目幼虫的能力。     

分离筛选大熊猫正常生理菌—乳杆菌

本实验从１７只健康大熊猫粪便中分离出４７株待检菌,通过对其菌体形态和染色性,菌落形态,生化反应,发酵最终产物的气相色谱测定鉴定筛先出１３株乳杆菌。并测定了１３株乳杆菌对大熊猫常用抗生素的药物敏感性和对小白鼠的急性毒力实验。对大熊猫正常生理菌群微生态制剂的研制进行了初步探讨     

用原生质体融合技术选育2—酮基—L—古龙酸高产菌株的研究

对革兰氏阳性的地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）Ｈ１９和革兰氏阴性的２－酮基－Ｌ－古龙酸产生菌Ｓ１９的原生质体的制备条件进行了研究,并采用聚乙二醇作诱导剂进行了两菌株的原生质体融合,用链霉素作为抗性标记对融合子进行了选择。从１７株产生２－酮基－Ｌ－古龙酸的融合子中选出了一株连续传代八次产酸高且产量稳定的融合子１５号。融合子１５号具有两个亲本菌株所具有的一些特性。     

链霉菌菌株A与哈茨木霉T-23原生质体融合条件的研究

报道了链霉菌菌株和哈茨木霉菌株属问原生质体融合构建生防工程菌的前期研究成果,研究结果显示：链霉菌菌株A与哈茨木霉T-23分别以1000μg／ml庆大霉素和50～53℃热灭活120min作为遗传标记;融合系统采用聚乙二醇(PEG)作为促融剂,通过对PEG最佳分子量、浓度、处理时间的筛选,确立最佳融合技术系统,即内含0．05mol／L Ca^2 的35％ PEG6000,融合处理15min。所得融合子经过选择再生培养基培养后,在132株融合子中初步筛选出2株稳定的融合子。经孢子大小和DNA含量测定,确立一株为单倍重组体,另一株为杂合二倍体。     

Synthesis and analysis of a methyl ether derivative of tetracycline which inhibits growth of Escherichia coli

Tetracycline is a widely used broad spectrum antibiotic. A derivative of tetracycline was synthesized by methylation (-CH3) of the phenolic hydroxyl group, with the use of diazomethane (CH2N2). A methyl ether group is then formed from the reaction with diazomethane, which replaces the hydroxyl group. The newly formed derivative has reduced hydrogen bonding capability relative to the unmodified tetracycline. An infrared spectra shows the appearance of the ether group on the derivative and the Log P calculations indicate that the derivative has increased lipophilic tendency. The Lipophilic Substituent Constant calculated for the tetracycline derivative is 0.46, indicating a lipophilic substituent. The tetracycline derivative was soluble in aqueous solvents and was stable for more than five weeks when stored at < or = 0 degrees C. The derivative was placed in tissue culture utilizing Luria-Bertani (LB) media, at a concentration of 12.0 microg/mL and inhibited the growth of E. coli (XL-1 blue) from 15% to 20% within the initial sixteen hours.     

Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.     

Convenient preparation of [Orn(Tfa)2]- and [Orn(Boc)2, Orn(Tfa)2]gramicidin S, versatile unsymmetrically protected derivatives of gramicidin S.

Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS. The mono(Tfa) derivative [Orn(Tfa)2']GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative [Orn(Boc)2,Orn(Tfa)2']GS from which another monoprotected derivative [Orn(Boc)2]GS was prepared in high yield. These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives. As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative [Orn(Boc)2,Orn(NBS)2']GS.     

Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

The biological activities of C₆₀-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and -8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.     

Studies on beta-galactoside transport in a Proteus mirabilis merodiploid carrying an Escherichia coli lactose operon

Merodiploid derivatives bearing an F-linked lac operon (i(+), o(+), z(+), y(+), a(+)) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli. A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli. In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-beta-d-galactoside) were similar for both beta-galactosidase activity (beta-d-galactoside galactohydrolase, EC 3.4.1.23) and beta-galactoside transport activity in both derivatives, although the ratio of galactoside transport to beta-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of beta-galactosidase and M-protein (lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal beta-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o-nitrophenyl-beta-galactoside from an outside region of high concentration to an inside region of very low concentration ("downhill transport") was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-beta-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-beta-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.     

Synthesis of the alpha-D-GlcpA-(1-->3)-alpha-L-Rhap-(1-->2)-L-Rha trisaccharide isolated from the cell wall hydrolyzate of the green alga, Chlorella vulgaris

The title trisaccharide was synthesized from 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride (10), ethyl 2,4-di-O-benzyl-1-thio- (5) and benzyl 3,4-di-O-benzyl-alpha-L-rhamnopyranoside (9). The disaccharide 11 obtained from compounds 5 and 10 was used as the glycosyl donor to glycosylate the rhamnopyranoside derivative 9 having free OH-2 using the NIS-AgOTf-mediated glycosylation methodology. Zemplén deacetylation of the trisaccharide 12 resulted in the 6"-OH derivative (13), which was selectively oxidized with CrO3 to the uronic acid derivative 14. The benzyl groups were removed by catalytic hydrogenolysis to furnish the target trisaccharide (1).     

The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5′-GA dinucleotides and sequences that contained 5′-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.     

Synthetic study on carbocyclic analogs of cyclic ADP-ribose, a novel second messenger: an efficient synthesis of cyclic IDP-carbocyclic-ribose.

An efficient synthesis of cyclic IDP-carbocyclic-ribose, as a stable mimic for cyclic ADP-ribose, was achieved. 8-Bromo-N1-carbocyclic-ribosylinosine derivative 10, prepared from N1-(2,4-dinitrophenyl)inosine derivative 5 and an optically active carbocyclic amine 6, was converted to 8-bromo-N1-carbocyclic-ribosylinosine bisphosphate derivative 15. Treatment of 15 with I2 in the presence of molecular sieves in pyridine gave the desired cyclic product 16 quantitatively, which was deprotected and reductively debrominated to give the target cyclic IDP-carbocyclic-ribose (3).     

Behavior of hobo and P transposons in yellow2-717 unstable line of Drosophila melanogaster and its derivatives after crossing with a laboratory strain

Using fluorescent in situ hybridization technique (FISH), the frequency of hobo and P mobile elements transpositions on X chromosomes from the y2-717, isolated from the Uman' population of Drosophila melanogaster, as well as from its phenotypically normal and mutant derivatives, obtained as a result of crosses the males examined with the C(I)DX, ywf/Y females, was evaluated. It was demonstrated that the maximum frequency of hobo transpositions on X chromosomes of the males from derivative strains, subjected to repeated hobo-dysgenic crosses reached a value of 1.2 x 10(-2) per site per X chromosome per generation. The number of hobo copies in male X chromosomes from derivative strains was 3 times higher than in the original initial strain. Furthermore, the "old" hobo sites remained unchanged. In derivative strains, the frequency of hobo insertions was higher than that of excisions. One of the derivative strains, y1t-717alk3-2, was characterized by high intra-strain instability of hobo element localization. In the y2-717a1k3 and y1t-717alk3-2 strains a large inversion, In(1)1B; 13CD, was described. At the absence of the full-sized P element in the strains involved in crosses, maximum frequency of P element transpositions in the derivative strains reached a value of 1.2 x 10(-2) per site per X chromosome per generation.