利用弗氏链霉菌的修饰系统克服吸水链霉菌的限制性障碍——发展吸水链霉菌转

用来自变铅青链霉菌TK24的质粒PIJ702（tsr,mel^ )转化吸水链霉菌应城奕种10－22的原生质体,未能得到转化子,但改用来自弗氏链霉菌ATCC 10745的pIJ702转化10－22原生质体却得到了转化子,转化频率约10^3-10^4转化子／微克DNA,在这些转化子中,只有约1／1000是黑色菌落（mel^ ),绝大部分为白色菌落（mel^-),分别挑出黑色,白色2种菌落,只有从前者能提出电泳可见的PIJ702质粒带。2种菌落的质粒提取物,均能转化TK24原生质体并正常表达黑色素,在非选择性条件下,从10－22（PIJ702）黑色菌落的群体中,获得了少数白色菌落,经证明它们是10－22的宿主突变体,这些突变体的PIJ702转化子出现均一的黑菌落。     

利用弗氏链霉菌的修饰系统克服吸水链霉菌的限制性障碍——发展吸水链霉菌转化系统的尝试

用来自变铅青链霉菌TK24的质粒plJT02(tsr、mel~+)转化吸水链毒菌应城变种10-22的原生质体,未能得到转化子。但改用来自弗氏链霉菌ATCC 10745的plJ702转化10—22原生质体却得到了转化子。转化频率约10 ~3—10~4转化子/微克DNA。在这些转化子中,只有约1/1000是黑色菌落(mel~+),绝大部分为白色菌落(mel~-)。分别挑出黑色、白色2种菌落,只有从前者能提出电泳可见的plJ702质粒带。2种菌落的质粒提取物,均能转化TK24原生质体并正常表达黑色素。在非选择性条件下,从10-22(pIJ702)黑色菌落的群体中,获得了少数白色菌落,经证明它们是10-22的宿主突变体。这些突变体的plJ702转化子出现均一的黑色菌落。     

不吸水链霉菌梧州新亚种限制-修饰系统初探

采用菌丝体原位包埋方法和高压脉冲电泳技术从不吸水链霉菌梧州新亚种(Streptomyces ahygroscopicus wuzhouensis neosubsp. 11371)中分离得到两条质粒DNA带.通过双向电泳证明,2个质粒均为线性分子,按照分子量大小依次命名为pSAL1、pSAL2.并对不吸水链霉菌梧州新亚种的限制-修饰系统进行初步探讨:将来自变铅青链霉菌TK54的高拷贝质粒pIJ702转化不吸水链霉菌梧州新亚种原生质体,未能得到转化子,改用pIJ702转化不吸水链霉菌梧州新亚种U-3原生质体,得到了转化子.     

生米卡链霉菌变株丙酰化酶基因的克隆和表达

麦迪霉素产生菌生米卡链霉菌(streptomyces mycarofaciens)变株具有丙酰化酶活性,可以将螺旋霉素转化为丙酰螺旋霉素。为了进行丙酰化酶基因克隆,本实验以质粒pIJ702为载体通过鸟枪克隆法将变株DNA片段克隆至变铅青链霉菌TK54(Streptomyces livdansTK54),经薄层层析和高压液相色谱分析结果表明,在转化子中,N0．9菌株可以将螺旋霉素转化为丙酰螺旋霉素,这证明丙酰化酶基因已在变铅青链霉菌TK54中克隆并得到初步表达,№．9重组质粒插入DNA片段为4．16kb,经southern杂交表明确实来源于变株。此外还构建了N0．9重组质粒的限制酶酶切图谱。     

黑暗链霉菌DNA转移系统的建立

研究了黑暗链霉菌的基因转移系统,探索了通过PEG介导的原生质体转化、接合转移向黑暗链霉菌中转入外源DNA的可能性。多次尝试用质粒pIJ702转化黑暗链霉菌9904原生质体均未成功。对原生质体进行“热处理”后转化、利用单链DNA转化等都不能将质粒导入黑暗链霉菌中,表明黑暗链霉菌对外源DNA有很强的限制修饰作用。利用接合转移将具有oriT的大肠杆菌链霉菌穿梭质粒pHZ132转入大肠杆菌ET12567(pUZ8002)中,获得供体菌ET12567(pUZ8002,pHZ132)。将供体菌与预萌发的黑暗链霉菌9904的孢子进行接合转移,成功地将pHZ132转入黑暗链霉菌9904中。质粒pHZ132经黑暗链霉菌自身修饰后也可转入黑暗链霉菌9904菌株的原生质体中,转化率约为103/μg DNA(pHZ132)。     

大肠杆菌-链霉菌穿梭载体的构建及应用

pIJ6021和pIJ4123是链霉菌的高拷贝表达载体,它们携带有受硫链丝菌素诱导的强启动子P_tipA。分别在它们的合适位点插入大肠杆菌质粒的复制子和在大肠杆菌中选择用的抗性标记基因(bla),得到了两个能在大肠杆菌和链霉菌中穿梭复制、并保持结构稳定的链霉菌表达载体：pHZ1271和pHZ1272。将透明颤菌(Vitreoscillia sp.)血红蛋白基因(vhb)克隆到pHZ1272中,用它转化变铅青链霉菌(Streptomyces lividans),经Western blotting分析和CO结合实验表明,在变铅青链霉菌中表达出了有生物活性的透明颤菌血红蛋白,从而证明所构建的pHZ1272载体具有在链霉菌中表达外源基因的功能。     

一个枯草杆菌启动子(P_(28-1))对链霉菌分化的影响

亚克隆1.1kb的枯草杆菌启动子P_(28-1)到pUC19上,再亚克隆到以儿茶酚加氧酶为报告基因的链霉菌启动子探测质粒pIJ4083上,构建的重组质粒命名为pIJ4498.用pIJ4498转化天蓝色链霉菌J1501的原生质体,得到了相对于灰色野生型的白色转化子,而用载体pIJ4083转化J1501后得到的转化子是正常的深灰色菌落.经限制性内切酶验证了重组质粒的结构,测定了质粒的稳定性.当pIJ4498转化天蓝色链霉菌的WhiG突变株(C71)后,未观察到任何表型的变化.通过超声波破碎细胞得到的J1501/pIJ4498菌体的蛋白提取液,可使无色的儿茶酚氧化成黄色的2-羟粘糠酸半醛(HMS).而对照株J1501/pIJ4083及C71/pIJ4498菌株的蛋白提取液不能使儿茶酚氧化成黄色的HMS产物.结果表明枯草杆菌的启动子P_(28-1)被天蓝色链霉菌J1501的σ^(whiG) RNA聚合酶所识别,在启动儿茶酚加氧酶报告基因表达的同时,影响了天蓝色链霉菌J1501分化中的孢子形成.     

一个枯草杆菌启动子(P_(28-1))对链霉菌分化的影响

亚克隆1.1kb的枯草杆菌启动子P_(28-1)到pUC19上,再亚克隆到以儿茶酚加氧酶为报告基因的链霉菌启动子探测质粒pIJ4083上,构建的重组质粒命名为pIJ4498.用pIJ4498转化天蓝色链霉菌J1501的原生质体,得到了相对于灰色野生型的白色转化子,而用载体pIJ4083转化J1501后得到的转化子是正常的深灰色菌落.经限制性内切酶验证了重组质粒的结构,测定了质粒的稳定性.当pIJ4498转化天蓝色链霉菌的WhiG突变株(C71)后,未观察到任何表型的变化.通过超声波破碎细胞得到的J1501/pIJ4498菌体的蛋白提取液,可使无色的儿茶酚氧化成黄色的2-羟粘糠酸半醛(HMS).而对照株J1501/pIJ4083及C71/pIJ4498菌株的蛋白提取液不能使儿茶酚氧化成黄色的HMS产物.结果表明枯草杆菌的启动子P_(28-1)被天蓝色链霉菌J1501的σ~(whiG) RNA聚合酶所识别,在启动儿茶酚加氧酶报告基因表达的同时,影响了天蓝色链霉菌J1501分化中的孢子形成.     

人可溶性血管内皮细胞生长因子受体Flt-1基因在链霉菌中的克隆表达

应用RTPCR技术,从人脐静脉内皮细胞中扩增出编码人可溶性血管内皮细胞生长因子 (VEGF)受体Flt1胞外区前四个结构域的基因片段,亚克隆至pUCl8质粒进行测序,将目的基因片段连接至链霉菌表达载体pSGLgpp,获得重组质粒pSGLgppF,将其转化至Streptomyces lividans TK24, 获得基因工程菌株Sreptomyces lividans (pSGLgppF),对其培养上清液进行SDSPAGE及Western blot分析,结果 显示,在636kD处有特异性条带出现,表明sFLT1在链霉菌中获得了成功表达,受体配基结合实验显示表达产物与VEGF可特异性结合,表明其具有配基结合生物活性。     

Tn4560在圈卷产色链霉菌中的转座及其在尼可霉素生物合成相关基因克隆中的应用

采用常规转化方法用来自天蓝色链霉菌J1 5 0 1的质粒pUC1 1 6 9(pMT6 6 0∷Tn45 5 6∷vph)多次转化尼可霉素产生菌圈卷产色链霉菌野生型 71 0 0的原生质体 ,均未得到转化子。采用限制性热衰减法于 5 0℃ ,3 0min溶菌制备 71 0 0的原生质体 ,获得了转化子 ,但转化频率极低 ,只有 0 4个转化子 μgDNA。用来自 71 0 0的pUC1 1 6 9再转化不含pUC1 1 6 9的 71 0 0原生质体 ,转化频率提高 1 0 3 ～ 1 0 4 倍。于 3 9℃ ,MM Vio条件下培养携带有pUC1 1 6 9的 71 0 0孢子 ,Tn45 6 0发生转座 ,筛选到 40 6 8个转座菌落 ,并从中得到 8株尼可霉素阻断突变株 ;对这 8株突变株的总DNA进行Southern杂交分析表明 ,Tn45 6 0至少在 4个不同的位点插入到 71 0 0的染色体上。用实验室已获得的与尼可霉素生物合成有关的 3 0kbDNA片段为探针和经不同酶切的 8株突变株的总DNA进行Southern杂交 ,结果表明 ,除阻断突变株Nik5有杂交信号且杂交信号大小均同野生型…     

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.