酵母细胞的电击高频转化

用Ｂｉｏ－Ｒａｄ生产的基因脉冲仪进行酿酒酵母电击转化实验,得到的最适条件为：５ｋｖ／ｃｍ２５μＦ和２００Ω。电击后涂布前的培养时间为２小时。电击后细胞存活率为４６％时,每微克质粒ＤＮＡ得到１０６以上的转化子。用相同的质粒和受体菌进行原生质体法和醋酸锂法比较实验,转化率分别为２×１０４和３．５×１０２个转化子／μｇＤＮＡ。电击转化是最方便易行和高效率的方法。     

niaD基因与uidA基因共转化糖化酶高产菌株Aspergillus nigerT21

从Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ Ｔ２１分离到自发性的氯酸盐抗性株,再经氮源生长试验获得硝酸盐还原酶缺陷的ｎｉａＤ突变体Ｎ４４。用含有ｎｉａＤ的质粒ｐＳＴＡ１０转化Ｎ４４,转化频率为５个／μｇ（转化子／ＤＮＡ）。转化子的Ｓｏｕｔｈｅｒｎ印迹分析表明ｎｉａＤ基因同源整合到Ｎ４４的染色体ＤＮＡ中。ｐＳＴＡ１０与含葡萄苷酸酶基因（ｕｉｄＡ）的质粒ｐＮＯＭ１０２共转化Ｎ４４,共转化频率为４０％。共转化     

质粒pMC71A—LEU转化阴沟肠杆菌E26—1的研究

质粒pMC71A－LEU在对阴沟肠杆菌E26－1（EnterobactercloacaeE26－1）的转化中研究了感受态细胞、CaCl2浓度、热激温度和质粒DNA量对转化频率的影响。结果表明,在阴沟肠杆菌F26－1生长到OD600＝0．45时,用50mmol／LCaCl2处理细胞,加入100ng质粒DNA,在32℃热激2min,转化频率最高,可达1×103转化子／μgDNA。     

植病生防菌成团泛菌电击转化条件的研究*

研究了植病生防菌成团泛菌（Ｐａｎｔｏｅａ ａｇｇｌｏｍｅｒａｎｓ）的电击转化条件。结果表明受体菌固体培养、生长到对数早期时的细胞转化效果最好;质粒ＤＮＡ分子量增加１０培,转化效率降低１００倍;质粒ＤＮＡ浓度在０．０１－１μｇ／μＬ范围内其变化与转化频率呈正比,与转化效率成反比;从成团泛菌中提纯的质粒ＤＮＡ比从大肠杆菌中提纯的相同质粒ＤＮＡ转化成团泛菌的效率高出３０倍;最佳电击条件为场强１５ｋＶ／ｃ     

具有真细菌基因启动子活性的盐生盐杆菌质粒DNA片段

利用大肠杆菌启动子探测质粒ｐＫＫ２３２－８为载体、用两组限制性内切酶ＢａｍＨＩ－ＳａｌＩ和ＨｉｎｄⅢ－ＳａｌＩ分别消化盐生盐杆菌Ｊ７（Ｈａｌｏｂａｃｔｅｒｉｕｍｈａｌｏｂｉｕｍ）的质粒ｐＨＨ２０５,在体外进行重组,转化Ｅ．ｃｏｌｉＨＢ１０１感受态细胞,在含氨苄青霉素和氯霉素的选择平板上筛选转化子,并从随机挑选的２０株转化子中,获得抗氯霉素水平达到１１０μｇ／ｍｌ的转化子Ｔ１和Ｔ２,所含重组质粒分别被命名为ｐＪＨ和ｐＪＢ。经限制性酶切分析及杂交分析表明,ｐＪＨ质粒上插入了一段来源于ｐＨＨ２０５质粒的ＤＮＡ片段,其大小为８００ｂｐ左右。通过重新转化实验进一步表明,该ＤＮＡ片段在大肠杆菌中具有启动子功能,从而证明,在古细菌（盐生盐杆菌）的质粒ＤＮＡ中存在具有真细菌（大肠杆菌）基因启动子活性的ＤＮＡ片段。     

以潮霉素抗性为选择标记的稻瘟病菌原生质体转化

稻瘟病菌对潮霉素（ＨｙｇｒｏｍｙｃｉｎＢ,简写为ＨｍＢ）较为敏感,当不含无机盐的再生培养基中ＨｍＢ浓度达到１００μｇ／ｍｌ时,稻瘟病菌２５３９ｗ原生质体的再生即被完全抑制。我们利用带有ＨｍＢ抗性基因的质粒（ｐＡＮ７－１）,通过ＰＥＧ②融合法对其原生质体进行转化,获得了ＨｍＢ抗性转化子。转化子以两类形式出现：一类为大而生长稳定的菌落,另一类为小而生长不稳定的菌落,两类菌落产生频率分别为每μｇＤＮＡ３－４个和１０－２０个。将大菌落转化子转接到含有ＨｍＢ的新培养基上,仍可正常生长,但小菌落转化子却不能,说明前者为真正的转化子而后者为流产转化子。ＤＮＡ杂交分析显示转化是由于质粒ＤＮＡ整合到了２５３９ｗ的染色体ＤＮＡ上,整合可在染色体ＤＮＡ的不同位点上发生。受试的７个转化子尽管各自的整合形式不同,但均在选择与非选择性培养中保持稳定。     

PEG法介导转化诸葛菜下胚轴原生质体获得转基因植株

采用诸葛菜无菌苗的下胚轴组织为材料,分离原生质体,在原生质体培养基中作液体浅层暗培养,植板率为５％,植株再生频率为１００％。作者进而开展了遗传转化研究。为研究ＰＥＧ介导转化诸葛菜原生质体的影响因素,通过瞬间表达,实验了ＰＥＧ法转化子叶原生质体的过程,在此基础上将分离纯化后的原生质体与带ＨＰＴ基因的质粒ＤＮＡ（ｐＢＩ２２２）混合,ＨＰＴ基因作选择标记,ＰＥＧ介导转化;重新收集转化后的原生质体,以５×１０４／ｍｌ的密度在原生质体培养基中作浅层培养;培养１０—１５天后用２５ｍｇ／Ｌ的潮霉素（ｈｙｇｒｏｍｙｃｉｎ）进行筛选,一月后出现少量细胞团,转入含潮霉素５０ｍｇ／Ｌ的扩增培养基扩增愈伤组织,进而转入含５０—１００ｍｇ／Ｌ潮霉素的分化培养基诱导分化成苗,分化率为１００％,转入生根培养基中生根成完整植株。抗性植株再生率为４×１０（－５）。在获得再生转基因植株后,以再生植株叶片为材料,进行Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交,证实外源基因已稳定整合到植物基因组中并表达,再生转基因植株频率为１０（－５）。国内外首次转化诸葛菜属植物原生质体获得成功。     

同一质粒转入感受态细胞的四种方法转化效率的比较研究

同一质粒转入感受态细胞的四种方法转化效率的比较研究周国林付士红梁国栋（中国预防科学院病毒研究所,基因工程国家重点实验室,北京１０００５２）分类号Ｑ２转化特指以质粒ＤＮＡ或以它为载体构建的重组子导入细菌的过程。细菌细胞处于容易吸收外源ＤＮＡ的状态称为感...     

电转化法提高平连载体DNA转化效率的研究

探讨了电转化过程中一些影响平连载体ＤＮＡ转化效率的因素,并与化学法进行了比较,由此建立了优化的电转化条件。结果显示,在ＯＤ６００值０．７２－０．７８收获细胞时,可使平连载体ＤＮＡ的转化效率达到５×１０６转化子／μｇｃＤ－ＮＡ,较化学法高１０２－１０３倍。同时,降低连接产物的盐浓度,对于电转化成功及提高转化率也至关重要     

转化条件对质粒DNA转化大肠杆菌的影响

研究了质粒DNA大小、质粒DNA浓度、CaCl2 浓度、热休克时间及感受态细胞保藏时间等因素对大肠杆菌HB1 0 1和JM1 0 5转化频率的影响 ,并对转化子中质粒DNA进行了分离、酶切、琼脂糖凝胶电泳检测。结果表明 ,CaCl2 浓度、质粒大小和浓度 ,以及感受态细胞的活力对转化频率有重要影响 ,42℃热休克处理可以提高转化频率。     

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.