栖热菌的DNA转化研究

由栖热菌ＦＤＢ８提取的染色体ＤＮＡ对栖热菌ＦＤ３００９进行了转化。用丝裂霉素Ｃ选择培养基对转化子进行选择，共获得３个转化子。它们的菌落色泽、对丝裂霉素Ｃ的抗性强和耐热ＤＮＡ聚合酶活性均介于供体菌和受体菌之间，而其菌体生长速度和破壁难易程度则优于供体菌和受体菌。结果表明，转化子是由供体菌ＦＤＢ８的染色体ＤＮＡ转化了受体菌ＦＤ３００９所致，同时还可由转化子进一步选育出抗丝裂霉素Ｃ的耐热ＤＮＡ聚合酶高产     

酵母菌呼吸缺陷型和野生型线粒体DNA比较

以酒精酵母，ＩＦＦＩ１３００ 材料，建立了以”蜗牛酶＋机械振荡“复俣自理破碎细胞的方法；先提取并纯化线粒体，后从线粒体中提取线粒体ＤＮＡ的方法，得到了紫外吸收Ａ２６０／２８０为１．８２３，琼脂糖电泳为一条带且分子量大λＤＮＡ的线粒体ＤＮＡ。     

人工染色体研究进展

细菌人工染色体、噬菌体Ｐ１衍生人的人工染色体和酵母人工染色体是近年发展起来的ＤＮＡ克隆新技术。着重介绍了ＹＡＣｓ作为转移大分子外源ＤＮＡ的载体，在生物基因组分析析基因的结构与功能、表达与调控、定位与分离以及遗传病的基因治疗等研究领域的应用。比较了ＢＡＣｓ、ＰＡＣｓ和ＹＡＣｓ的主要特点。对哺乳动物人工染色体的研究情况也作了简要介绍。     

对在酵母V号染色体克隆ARS过程中发现的一…

早期在有关Ａ３６４ａ的Ｖ号染色体ＡＲＳ研究中发现３株再次重组的含ＡＲＳ的质粒。在深入探讨这一再次重组现象的机理时，发现它们的外源片段能与Ａ３６４ａ的Ｖ号染色体ＤＮＡ杂交，而不与大肠杆菌ＤＮＡ或酵母转座子杂交。证明这个外源片段来源于酵母Ｖ号染色体，而不来自Ａ３６４ａ其他染色体（因为它来自Ｖ号染色体专一文库）。并意外地发现它与Ｃａｒ２基因５＇调控区高度同源（９５％），与其编码区也有７３．４％同源。提出     

转座子K1.4可提高荧光素酶基因在转基因家蚕中的整合 …

合适的整合表达载体对于获得转基因家蚕具有重要意义,为此本文构建了包含家蚕转座子Ｋ１．４,丝素基因上游启动子序列和荧光素酶基因,ｃＤＮＡ的质粒ＳＫＦＬ以及不含Ｋ１．４的质粒ｐＦＬ,并将其通过扎卵法转移到家蚕受精卵中,通过对当代转基因家蚕５龄幼虫丝腺和蚕体ＤＮＡ进行斑点杂交和Ｓｏｕｔｈｅｒｎ杂交发现,荧光素酶基因在家蚕不同组织中的整合是随机的,但携带Ｋ１．４的质粒ＳＫＦＬ的整合频率明显高于质粒ｐＦＬ。     

人工染色体研究进展

细菌人工染色体（ＢＡＣｓ）、噬菌体Ｐ１衍生的人工染色体（ＢＡＣｓ）和酵母人工染色体（ＹＡＣｓ）是近年发展起来的ＤＮＡ克隆新技术。着重介绍了ＹＡＣｓ作为转移大分子外源ＤＮＡ的载体，在生物基因组分析，基因的结构与功能、表达与调控、定位与分离以及遗传病的基固治疗等研究领域的应用。比较了ＢＡＣｓ、ＰＡＣｓ和ＹＡＣｓ的主要特点。对哺乳动物人工染色体的研究情况也作了简要介绍。     

性别鉴定的分子生物学技术与ZFY途径

立足性别决定的雄性决定说，综述了三十年来搜寻Ｙ染色体上睾丸决定因子（ＴＤＦ）的进程以及在此理论基础上发展起来的Ｙ－特异ＤＮＡ探针杂交、ＰＣＲ扩增Ｙ－特异ＤＮＡ、ＰＣＲ扩增ＳＲＹ序列、ＰＣＲ扩增ＺＦＹ序列等分子水平性别鉴定技术；比较了各种技术的长处与不足以及采用ＺＦＹ序列进行性别鉴定的优势     

转座子K1.4可提高荧光素酶基因在转基因家蚕中的整合频率

合适的整合表达载体对于获得转基因家蚕具有重要意义,为此本文构建了包含家蚕转座子Ｋ１．４、丝素基因上游启动子序列和荧光素酶基因ｃＤＮＡ的质粒ＳＫＦＬ以及不含Ｋ１．４的质粒ｐＦＬ,并将其通过扎卵法转移到家蚕受精卵中．通过对当代转基因家蚕５龄幼虫丝腺和蚕体ＤＮＡ进行斑点杂交和Ｓｏｕｔｈｅｒｎ杂交发现,荧光素酶基因在家蚕不同组织中的整合是随机的,但携带Ｋ１．４的质粒ＳＫＦＬ的整合频率明显高于质粒ｐＦＬ．这说明转座子Ｋ１．４可以有效提高外源基因在转基因家蚕中的整合频率,从而有可能成为构建转基因家蚕的一种有用的整合载体．     

跨界原生质体融合产物细胞遗传物质整合过程中DNA含量变化

报导光合细菌球形红假单胞菌与酿酒酵母跨界原生质体融合的最初产物细胞Ｆ０和Ｆ０ａ在连续９６小时的培养过程中细胞ＤＮＡ含量的变化规律．酵母及光合细菌ＤＮＡ含量分别为（４．２１±０．０４）×１０－８μｇ／ｃｅｌ和（２．４４±０．１２）×１０－８μｇ／ｃｅｌ．Ｆ０培养２４小时ＤＮＡ含量为（１０．５±０．５９）×１０－８μｇ／ｃｅｌ，到培养９６小时为（５．３０±０．４０）×１０－８μｇ／ｃｅｌ，高于任一亲株；而Ｆ０ａ则从（１７．１±０．５９）×１０－８μｇ／ｃｅｌ降低至（９．２７±０．３７）×１０－８μｇ／ｃｅｌ，高于双亲ＤＮＡ含量之和．经统计学ｔ检验，融合产物ＤＮＡ含量与双亲ＤＮＡ含量均存在显著差异ｔ＞ｔ０．０１（１０）．本研究表明原生质体融合初期，遗传物质ＤＮＡ在整合过程中数量逐步降低后可达稳定水平，而其含量高于任一亲株细胞的基本规律．     

β—内酰胺酶基因在谷氨酸产生菌T6—13染色体上的…

以质粒ｐＵＣ１３编码的β－内酰胺酶基因为外源基因，通过随机连接谷氨酸产生菌Ｔ６－１３染色体ＤＮＡ片段后转化Ｔ６－１３原生质体，使其整合到染色体上，得以表达。在实验条件下，所测１９株转化子的抗性都有很高的稳定性，其中１０株能百分之百地保持抗性。经生物素标记的ｐＵＣ１３为探针的分子杂交实验证实抗性基因已整合到Ｔ６－１３菌染色体ＤＮＡ上。     

Expression of a bacterial modification methylase gene in yeast

Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not to contain 5-methylcytosine in their DNA. It would be interesting to test, how gene expression in such organisms would respond to the methylation of specific cytosines in the genome. As a first step towards this goal, we have introduced the gene encoding the Bacillus sphaericus R modification methylase, which methylates the internal cytosine within the recognition sequence 5'-GGCC, into yeast cells. Southern-type hybridization to DNAs isolated from the transformed yeast clones revealed that the yeast plasmid carrying the prokaryotic methylase gene, as well as the two chromosomal genes tested (his3 and leu2) were methylated, whereas the bulk of the yeast DNA remained largely unmethylated. This indicates that the Bacillus sphaericus modification methylase was expressed in yeast but it modified only certain parts of the yeast DNA.     

抗菌肽SMAP-29在毕赤酵母中的表达

依据毕赤酵母密码子偏好性,设计合成抗菌肽SMAP-29成熟肽基因片段,克隆到表达载体pPIC3.5K上,SalI线性化后转化毕赤酵母GS115,418抗性筛选高拷贝克隆,再由酵母菌落PCR鉴定;阳性克隆用甲醇诱导表达,Tricine-SDS-PAGE分析,结果在诱导第2d的酵母裂解液中检测到与预期的SMAP-29分子量接近,约3.2kD的诱导表达带;Trizol法提取酵母总RNA,并通过RT-PCR扩增SMAP-29mRNA,发现表达期的酵母细胞中存在SMAP-29mRNA,而对照没有检出,表明SMAP-29在毕赤酵母中存在表达。     

Transgenes as probes for active chromosomal domains in mouse development

Embryonic development entails a well defined temporal and spatial programme of gene expression, which may be influenced by active chromosomal domains. These chromosomal domains can be detected using transgenes which integrate randomly throughout the genome, as their expression can be affected by chromosomal position. Position effects are probably exerted most strongly on transgenes that do not contain strong promoters, enhancers or other modulating sequences. Here we have systematically explored position effects using a transgene with the weak herpes-simplex-virus thymidine-kinase promoter, linked to the readily visualized lacZ indicator gene (HSV-TK-lacZ). Each transgenic fetus with detectable expression displayed a unique lacZ staining pattern. Thus expression of this construct is apparently dictated entirely by its chromosomal position, without any construct specificity. Furthermore the transgene is faithfully transmitted to subsequent generations, allowing for systematic mapping of changes in expression during development and in adult life. These results demonstrate that transgenes can indeed be powerful tools to probe the genome for active chromosomal regions, with the potential for identifying endogenous genes involved in organogenesis and pattern formation.     

Chromosomal evolution in Saccharomyces

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.     

Gene conversion between duplicated genetic elements in yeast

The mitotic recombination behaviour of a duplication of the his4 region on chromosome III in the yeast Saccharomyces cerevisiae was studied. The major recombination event between the duplicated segments is gene conversion unassociated with reciprocal recombination. The rad52-1 mutation preferentially decreases mitotic gene conversion. These results suggest that mitotic gene conversion may occur by a different pathway from that occurring in meiosis. This mitotic gene conversion may be important in yeast mating type interconversion and the maintenance of sequence homogeneity in families of repeated eukaryotic genes.     

DNA transformation leads to pilin antigenic variation in Neisseria gonorrhoeae

Many pathogenic bacteria express pili (fimbriae) on their cell surfaces. These structures mediate binding of bacteria to host tissues, and may also be involved in other aspects of pathogenesis. Neisseria gonorrhoeae pili are mainly composed of a single protein, pilin, whose expression is controlled at chromosomal expression loci (pilE). An intact pilin gene and promoter sequences are only found at pilE. Strain MS11 contains two expression sites (pilE1 and pilE2), whereas several of its derivatives and other clinical isolates contain only one. Silent pilin loci (pilS1-pilS7) contain truncated variant pilin genes lacking the promoter and conserved pilin gene sequences. Pilin antigenic variation in N. gonorrhoeae occurs by DNA recombination between one of he silent partial variant gene segments in pilS and an expressed pilin gene in pilE. The recombination reactions are nonreciprocal, and therefore the mechanism has been classified as gene conversion. We report that much of the recombination between pilin loci actually occurs after transformation of living piliated cells by DNA liberated from lysed cells within a population. This constitutes a new molecular mechanism for an antigenic variation system, as well as the first specific function for a DNA transformation system.     

rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用

根据酵母整合质粒的设计要求,PCR扩增特定的2．2kb rDNA片段,并以此替换酿酒酵母(Saccharomyces cereristae)整合载体YIp5的URA3片段;在此基础上,引入G418抗性基因KanMX和酵母磷酸甘油激酶(phosphoglycerale kinase,PGK)组成型强启动子和终止子序列(PGKp-t),构建适合酿酒酵母工业菌株高拷贝整合表达载体pYMIKP,以细菌木糖异构酶(xylose isomerase,XI)基因xy/A为目标基因,通过载体pYMIKP引入到酵母工业菌株NAN-27中,酵母转化子在非选择培养条件下,连续生长50世代质粒稳定性为99．72％,目标基因高拷贝重组菌的木糖异构酶比酶活是对照菌株的67．2倍,达到0．672U／mg蛋白,实现了外源基因在酿酒酵母工业菌株中的稳定高效表达。