酿酒酵母超氧物歧化酶(SOD)基因的克隆和表达

通过PCR扩增技术从酿酒酵母中得到了Cu,zn—SOD的结构基因,此基因被亚克隆到大肠杆菌质粒载体pT7—7．得到重组质粒pT7－7：SOD。利用EcoRI和Pstl酶切pT7-7::SOD质粒．经琼脂糖凝腔电泳,DEAE-滤膜回收Cu。zn—SOD结构基因片段,将其亚克隆到M13中．并转化大肠杆菌,得到了重组质粒M13-::t SOD,酶切和纯化后的SOD基因,定向克隆到酵母质粒载体pHz－8的smal和EcoRI位点上,构建成重组质粒pHZ－8－l。经转化酵母受体菌ZH-l和DP—l后得到了转化子．来自于ZH—l的转化子在非选择性条件下培养40世代后仍有95％以上细胞保留重组质粒。而来自于DP-1的转化子很不稳定。经蛋白提取、聚丙烯酰胺凝胶电泳和酶活性测定结果表明,来自于zH－1转化子中SOD的表达量约为细胞可溶性蛋白的15％．并具有生物活性。     

Structure and function ofsawB, a gene involved in differentiation ofStreptomyces ansochromogenes

A partial DNA library of Streptomyces ansochromogenes 7100 was constructed by using plasmid plJ702 as vector and white mutant W19 as recipient. About 3 000 clones were obtained, two of which gave rise to the grey phenotype as wild type 7100. The plasmids were isolated from two transformants. The result indicated that the 5.2 kb and 5.8 kb DNA fragments were inserted into plJ702. The resulting recombinant plasmids were designated as pNL-1 and pNL-2 respectively. The 1.25 kb Pstl l-Apa l DNA fragment from pNL-1 was recognized as its complementarity to W19 strain. The nucleotide sequence of the 3.0 kb Pst I DNA fragment including 1.25 kb was determined and analyzed. The result indicated that this DNA fragment contains one complete open reading frame (ORF1) which encodes a protein with 295 amino acid residues, and this gene was designated as sawB. The deduced protein has 81% amino acid identities in comparison with that encoded by whiH in Streptomyces coelicolor. The function of sawB gene was studied by usi     

链霉菌的可转座因子

可转座因子是细菌遗传分析和分子操作十分有用的工具 ,它包括插入序列、转座子和转座噬菌体。细菌的可转座因子多数来自于革兰氏阴性菌 ,少数来自于革兰氏阳性菌。链霉菌是一类重要的革兰氏阳性菌 ,近年来 ,已发现了几种链霉菌的可转座因子 ,并对部分可转座因子的转座特征作了详细的研究。最早发现的链霉菌可转座因子是天蓝色链霉菌 (Ｓ .ｃｏｅｌｉｃｏｌｏｒ)Ａ3( 2 )中 1 6ｋｂ的插入片断ＩＳ1 1 0 [1 ] ,后来在白色链霉菌 (Ｓ .ａｌｂｕｓ)中发现了ＩＳ1 1 2 [2 ] ,在带棒链霉菌 (Ｓ .ｃｌａｖｕｌｉｇｅｒｕｓ)中发现了ＩＳ1 1 6[3] ,…     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

塔里木河中下游湿地及周边植物群落与环境因子的关系初探

选取塔里木河中下游湿地及周边16个典型植物群落样地调查和环境因子数据,采用主分量分析(PCA)排序技术和回归分析,定量分析湿地及周边植物群落在空间上的分布格局,以及群落结构特征和环境梯度之间的关系.结果表明,影响塔里木河中下游湿地及周边植物群落分布的第1主分量中,土壤水分和盐分影响最大,贡献率为35.70％;在第2主分量中,土壤养分的影响最大,贡献率为25.97％.植物群落分布的生境可分为沼生轻盐中营养生境、湿生中盐中营养生境、中生中盐低营养生境和中旱生重盐低营养生境4种类型.沿不同生境依次分布着沼泽植被、草甸植被、河岸疏林和盐生荒漠 盐化灌丛植被.塔里木河中下游湿地及周边植物群落的生态优势度与土壤水分和盐分复合梯度呈显著的一元线性相关.二元回归分析结果显示,塔里木河中下游湿地及周边土壤水分和盐分复合梯度与多样性指数和生态优势度二元指标呈极显著相关.     

Cloning,reassembling and integration of the entire nikkomycin biosynthetic gene cluster into <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis> lead to an improved nikkomycin production

Background  

Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials.     

Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA

Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

SanJ, an ATP-dependent picolinate-CoA ligase, catalyzes the conversion of picolinate to picolinate-CoA during nikkomycin biosynthesis in Streptomyces ansochromogenes

Nikkomycins, a group of peptidyl nucleoside antibiotics, are competitive inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned previously from Streptomyces ansochromogenes. The cluster contains 25 complete ORFs including sanJ. The sanJ gene was inactivated by the insertion of a kanamycin resistance gene and the resulting disruption mutants failed to produce nikkomycins. Moreover, the nikkomycin production was restored by complementation with a single copy of sanJ. The deduced product of sanJ bears striking sequence similarity with enzymes belonging to the adenylate-forming superfamily. sanJ was overexpressed as a His6-tagged fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography. The purified SanJ demonstrated adenylate ligase activity in the presence of picolinate or its analogs (benzoate, nicotinate, 4-methoxybenzoate, 4-hydroxybenzoate), ATP and Mg2+. SanJ was also found to catalyze the conversion of picolinate, benzoate, nicotinate to their corresponding CoA esters and 4-methoxybenzoate, 4-hydroxybenzoate to their respective AMP derivatives in vitro. This was unambiguously shown by using HPLC and electrospray ionization mass spectrometry (ESI-MS) or by comparing the reaction product with an authentic standard of benzoyl-CoA. These results indicated that sanJ encodes an ATP-dependent picolinate-CoA ligase which is essential for nikkomycin biosynthesis.