野油菜黄单胞菌S-152遗传转移系统的建立及外源性内切葡聚糖酶基因在其体内的表达

(通过细胞接合的方式将广宿主质粒pRK404、pKT230和RP4分别转入野油菜黄单胞菌S-152中,其转移频率均在10-4数量级上,从而建立起了S-152的转移系统。用同法将由载体质粒pRK404和白纹黄单胞菌XA1-1的CMC酶基因构成的重组质粒pND82转入到S-152中并得到了表达,使S-152(pND82)的胞内CMC酶酶活比出发菌株S-152提高了1倍。The wide-host-range plasmid vectors pRK404 and pKT230 were transferred into a recipient strain S-152 respectively by conjugation with the aid of the helper plasmid pRK2013 at frequencies of 10-4, and a genetic transfer system for S-152 was developed. A recombinant plasmid pND82, constructed by combining the plasmid vector pRK404 with a chromosomal DNA fragment coding for CMCase from XA1-1, was introduced into S-152 by this transfer system.As a result, the intracellular CMCase activity of S-152 (pND82) increases 1 time in comparison with S-152.     

野油菜黄单胞菌S-152内切葡聚糖酶基因在大肠杆菌中的克隆与表达

以质粒pUC9为载体,大肠杆菌JM83为宿主菌,用鸟枪法克隆到纤维素降解细菌野油菜黄单胞菌S-152的内切葡聚糖酶(CMCase)基因,克隆到的CMCas e基因位于2.7kb的HindⅢ片段上,该重组质粒命名为pUC9H-1。Southern印迹杂交分析结果显示所克隆到的内切葡聚糖酶基因与野油菜黄单胞菌染色体DNA有亲和性。对克隆株的酶学性质分析表明,其CMCase活力为0.310μmol Glu/mg蛋白·分钟,最适作用pH为6.4,最适作用温度为55℃。 Abstract:Using plasmid pUC9 as vector,Escherichia coli JM83 as host strain,an endoglucanase(CMCase)gene has been cloned by Shoot-gun method from cellulose-degrading bacteria Xanthomonas campestris S-152,The recombinant plasmid pCU9H-1 was isolated from the positive transformant producing CMCase.The CMCase gene located in a 2.7kb HindIII DNA fragment.The result of Southern hybridization indicated that the CMCase gene cloned has affinity to chromosome DNA of Xanthomonas campestris S-152.The analysis of enzymatic activity of CMCase positive clone JM83(pUC9H-1)was carried out and the result indicated that the CMCase activity was about 0.310μmol Glu/mg pro·min,the optimum pH was 6.4 and the optimum temperature was 55℃.     

野油菜黄单胞菌S—152遗传转移系统的建立及外源性内切葡聚糖酶基因在其体内的表达

通过细胞接合的方式将广宿主质粒pRK404、pKT230和RP4分别转入野油菜黄单胞菌S-152中,其转移频率均在10-4数量级上,从而建立起了S—152的转移系统．用同法将由载体质粒pRK404和白纹黄单胞菌XAI-1的CMC酶基因构成的重组质粒pND82转入到S－152中共得到了表达,使S—152（pND82）的胞内CMC酶酶活比出发菌株S-152提高了1倍．     

Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, te-loRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ e     

黄单胞菌(Xanthomonas campestris)XA5-5β-葡萄糖苷酶基因的克隆与表达

以黄单胞菌(Xanthomonas campestris)XA5-5为供体菌,广泛寄主质粒pRK404为载体,在大肠杆菌中克隆了一个β-葡萄糖苷酶基因,重组质粒pLZS1外源片段为1.1kb,将pLZS1接合导入黄单胞菌XA5-5,得到了克隆子XA5-5(pLZS1).以水杨苷为底物,XA5-5(pLZS1)的酶活性大大高于E.Coli JM83(pLZS1).并且质粒在XA5-5中能相对稳定地存在.初步结果表明,所克隆的基因编码的产物对于水杨苷底物有较强的亲和力,并可以在一定程度上降低XA5-5中酶与pNPG底物的亲和力,使其酶活减小.     

东方肉座菌EU7-22纤维素酶基因的克隆及序列分析

东方肉座菌EU7-22与XC-9、里氏木霉、康宁木霉、黑曲霉、斜卧青霉进行产纤维素酶比较,结果表明菌株EU7-22具有较高的产纤维素酶能力及完整的纤维素酶系.根据里氏木霉和绿色木霉的外切葡聚糖酶,内切葡聚糖酶及β-葡萄糖苷酶相关基因序列,设计引物PCR扩增出菌株EU7-22 cbhⅠ、cbhⅡ、egⅠ、egⅡ及bgl Ⅰ.基因序列经NCBI Blast分析表明,cbhⅠ与绿色木霉cbh1基因(FJ871063)同源性最高达99％;cbhⅡ与康宁木霉cbh2基因(DQ504304)同源性最高达99％;eg Ⅰ与长枝木霉egl基因(GU144298)同源性最高达99％;egⅡ与绿色木霉eg2基因(EF602036)同源性最高达99％;bglⅠ与菌株Trichoderma sp.SSL bgl基因(FJ040193)同源性最高达100％.5种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高.对上述纤维素酶基因编码的相应蛋白的分子量、等电点、N-糖基化位点、信号肽序列进行分析;对纤维素结合区及糖基水解酶家族特征结构区进行了定位;用SWISS-Model模拟了酶蛋白的三级结构.     

黄单胞菌(Xanthomonas campestris)XA5-5β-葡萄糖苷酶基因的克隆与表达

以黄单胞菌(Xanthomonas campestris)XA5-5为供体菌,广泛寄主质粒pRK404为载体,在大肠杆菌中克隆了一个β-葡萄糖苷酶基因,重组质粒pLZS1外源片段为1.1kb,将pLZS1接合导入黄单胞菌XA5-5,得到了克隆子XA5-5(pLZS1).以水杨苷为底物,XA5-5(pLZS1)的酶活性大大高于E.Coli JM83(pLZS1).并且质粒在XA5-5中能相对稳定地存在.初步结果表明,所克隆的基因编码的产物对于水杨苷底物有较强的亲和力,并可以在一定程度上降低XA5-5中酶与pNPG底物的亲和力,使其酶活减小.     

用电脉冲法将外源质粒DNA导入花生根瘤菌的研究

利用基因电转移仪(Gene Pulser^TM|),对快、慢生型花生根瘤菌85-7和1 47-3的电转化条件进行了系统的研究。结果表明:在对数中期(ABC~600|为0.6-0.7)收获的细胞在最高场强(12.5kV/cm)和短脉冲时间(2.5-5.0m sec)时达到最高转化效率(10 E5转化子/μgDNA)。转化子数随DNA终浓度在一定范围(26.24pg-1.5μg/ml)内呈线性增加, 随即表现出“饱和效应”。增加受体菌细胞浓度,能提高电转化效率;而质粒分子量的增加(11.9 -166kb)却使电转化效率降低。来自受体菌自身的同源质粒,因克服了宿主的限制-修饰性,可以极显著地提高电转化效率。电脉冲处理对受体菌自发突变没有影响。 Abstract:A systematic study of transformation conditions of peanut rhizobia 85-7 and 147-3 was conducted by using Bio-Rad Gene Pulser equipment.It was revealed that the highest transformation efficiency(105 transformants/μgDNA) was obtained from cells harvested at mid-log phase of ABC600 on 0.6-0.7 under the highest field strength(12.5kV/cm)and a short pulse length(2.5-5.0 msec).A linear increase of transformants was observed when DNA concentration was increased in the range of 26.24pg-1.5μg/ml and it became saturation afterwards.Transformation efficiency was also increased with the raise of recipient cell concentrations,but decreased with the increase of plasmid sizes from 11.9 to 166kb.A significant increase of transformation efficiency was revealed with the homologous plasmid isolated from fecipient itself since the effects of host restriction and modification were avoided.No significant effect of electroporation on spontaneous mutation was observed.     

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbhl gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbhl promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.