猪─仓鼠杂种细胞中猪染色体鉴定

对猪与仓鼠细胞融合形成的一稳定的杂种细胞系进行染色体分析,Ｇ１１ 染色显示该杂种细胞中具有１ 条猪的染色体和２１ 条仓鼠染色体,经ＰＣＲ 检测确证该杂种细胞中含有猪１２ 号染色体     

Human chromosome 12 is required for elevated HIV-1 expression in human-hamster hybrid cells

Host cell factors act together with regulatory genes of the human immunodeficiency virus (HIV) to control virus production. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV gene expression. DNA transfection experiments showed that 4 of 18 clones had high levels of HIV gene expression measured by both extracellular virus production and transactivation of the HIV long terminal repeat in the presence of the trans-activator (tat) gene. Karyotype analyses revealed a 94% concordance (17/18) between human chromosome 12 and HIV gene expression. Other chromosomes had an 11 to 72% concordance with virus production.     

Chromosomal rearrangement generating a composite gene for a developmental transcription factor

Differential gene expression in the mother cell chamber of sporulating cells of Bacillus subtilis is determined in part by an RNA polymerase sigma factor called sigma K (or sigma 27). The sigma K factor was assigned as the product of the sporulation gene spoIVCB on the basis of the partial aminoterminal amino acid sequence of the purified protein. The spoIVCB gene is now shown to be a truncated gene capable of specifying only the amino terminal half of sigma K. The carboxyl terminal half is specified by another sporulation gene, spoIIIC, to which spoIVCB becomes joined inframe at an intermediate stage of sporulation by site-specific recombination within a 5-base pair repeated sequence. Juxtaposition of spoIVCB and spoIIIC need not be reversible in that the mother cell and its chromosome are discarded at the end of the developmental cycle. The rearrangement of chromosomal DNA could account for the presence of sigma K selectively in the mother cell and may be a precedent for the generation of cell type-specific regulatory proteins in other developmental systems where cells undergo terminal differentiation.     

Interferon production and action in mouse, hamster and somatic hybrid mouse-hamster cells

A hybrid mouse-hamster cell line was developed from a mouse cell line which produces a high titer of interferon and is sensitive to its action, and a hamster cell line which produces little interferon and is relatively insensitive to its action. Parental cell lines demonstrated complete species specificity with respect to interferon production and action. The hybrid cells produced interferon (or interferons) effective when tested on the mouse cell line and primary hamster cells; the hybrids were sensitive to the action of both mouse and hamster interferons. Hybrid cells produced ten times more hamster interferon than the parent hamster cell line and were eight times more sensitive to hamster interferon than the parent hamster cell line.     

同源重组探针在暂时转化的烟草原生质体内的重组和缺失

通过电激法(electroporation)将同源重组探针pBC 7导入烟草原生质体,再从转化的烟草原生质体中提取DNA,并将其转化到E.coli DH 1细胞.结果在E.codi DH 1细胞中获得了一种新的质粒分子,该质粒含有功能的neo基因,与重组探针pBC 7相比有较大的缺失、倒位,并出现了新的限制酶切部位,重组探针pBC 7的主要特征为含有Tn5的neo基因两个截短的不等部份,只有通过重排才可能产生完整的neo基因,这表明了暂时转化的原生质体在外源DNA整合以前具有这样的基因重排功能。     

Syrian hamster female protein: analysis of female protein primary structure and gene expression

The concentration in plasma of the female protein (FP) of the golden Syrian hamster is regulated by sex steroids and by mediators of the acute-phase response to tissue injury or inflammation. A complementary DNA (cDNA) clone corresponding to FP was isolated from a hamster liver cDNA library and used to determine the nucleotide sequence and derived amino acid sequence of native FP. The primary sequence of FP is 69 percent identical to human serum amyloid P component and 50 percent identical to human C-reactive protein. Evidence showed that sex-limited and acute-phase control of the FP gene is pretranslational. The FP protein is thus a useful model for investigating dual regulation of expression of a single gene.     

K88菌毛基因的克隆及ST表位序列在菌毛基因上的融合

[目的]为了建立用于细胞表面展示的载体系统。[方法]利用PCR技术,以大肠杆菌质粒为模板扩增K88菌毛操纵子的结构基因faeC~faeH,并克隆到pBR322质粒载体中。合成一段替换序列,在菌毛抗原基因的超变区引入酶切位点ApaI和NcoI,合成耐热肠度素STII表位编码序列,并引入菌毛抗原基因的超变区。[结果]经PCR鉴定和限制性内切酶酶切证明,重组质粒pBR-fae插入片断大小为6.6kb,与预期相符。核苷酸序列分析证明,所得faeC~faeH序列正确。PCR筛选和测序验证证明,构建了K88菌毛-耐热肠度素STII的融合基因。[结论]试验成功获得了重组质粒pBR-fae-ST。     

四膜虫基因打靶载体的构建及其在虫体内的转化

 【目的】构建一个适于嗜热四膜虫（Tetrahymena thermophila）的基因打靶载体，并分析转化四膜虫的特性。【方法】以嗜热四膜虫组蛋白H4-I基因作为启动子，利用H4-I基因的5′和3′侧翼区作为同源臂，并且保留H4-I基因编码区的起始密码子ATG和终止密码子TGA，中间嵌入巴龙霉素抗性标记基因neo，从而构建一个基因打靶载体。将该载体电击转化到嗜热四膜虫接合株体内，使其同源重组到四膜虫H4-I基因上。通过巴龙霉素抗性筛选、PCR扩增目的基因对抗性虫株进行鉴定。光学和电子显微镜观察抗性虫株形态变化。【结果】成功构建了一个适于嗜热四膜虫的基因打靶载体，将其电击转化到四膜虫体内，抗性虫株的生长速度明显高于普通四膜虫，显微镜观察抗性虫株形态变小，大约是正常形态的1/20～1/30，抗性虫株表面光滑，未见纤毛。【结论】利用基因打靶载体将neo基因定向整合到四膜虫H4-Ⅰ基因内部，为今后在嗜热四膜虫体内表达外源蛋白奠定了基础。     

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.     

杂合肽基因的合成及在大肠杆菌中的表达

将LfcinB15-Ma12杂合肽基因克隆到载体pET32a上,构建抗菌肽LfcinB15-Ma12杂合肽基因,并在大肠杆菌中表达融合蛋白。根据已报道的抗菌肽LfcinB和Magainin基因的氨基酸序列,推导出其cDNA序列,将两者连接为杂合基因并克隆到载体pET32a上,IPTG诱导表达。构建了LfcinB15-Ma12杂合肽基因重组质粒,经PCR扩增和DNA测序分析,成功构建了LfcinB15-Ma12杂合肽基因重组质粒,经IPTG诱导,成功表达了杂合肽LfcinB15-Ma12。这为利用基因工程表达其他抗菌肽奠定了基础。     

猪乙脑病毒分离株CSF.XZ2D在BHK21细胞上的传代培养及E基因序列分析

为了解河南省规模化猪场日本乙型脑炎病毒(JEV)流行株的特征及其遗传基因的稳定性,将猪乙脑病毒分离株CSF.XZ-2D在BHK-21细胞上进行连续传代培养,并对其E基因的遗传稳定性进行了研究。结果表明,经连续传代25次后病毒E基因趋于稳定,氨基酸位点E271(E→V)和E278(V→L)传代后发生稳定点突变。与JEV毒力相关的部分位点如E107、E138、E176、E177和E315遗传稳定性较高,经连续传代后均未发生突变,与SA14-14-2相应位点完全一致。但是,另外一些位点的遗传稳定性较差,如E279和E312分别出现K→M→K和K→R→K→R的反复突变。这些突变是否与JEV的宿主细胞适应性及毒力变化相关有待进一步研究。     

PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。     

利用体细胞杂种克隆板进行染色体区域定位的数据分析

利用猪×啮齿类体细胞杂种克隆板对本室新克隆和分离的猪胶质细胞原纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)基因进行染色体区域定位，对应用体细胞杂种克隆板进行染色体区域定位的试验数据统计的基本过程和方法进行了分析，研究结果表明猪GFAP基因定位于染色体12p11-(2/3p13)区域（P＜0.1%），并讨论了特殊试验数据的分析问题。     

BmNPV DNA解旋酶基因酵母双杂交诱饵载体的构建

为了筛选家蚕对BmNPV DNA解旋酶的受体基因,根据GenBank公布的BmNPV (T3株)的序列,设计引物对其DNA解旋酶基因的核心结构域进行PCR扩增,成功克隆该基因并利用双酶切亚克隆至酵母双杂交诱饵载体PGBKT7.实验证明重组诱饵载体不能自激活,可以作为诱饵进行蛋白结合试验.     

Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus

Simian acquired immune deficiency syndrome (SAIDS) in the macaque genus of monkeys at the California Primate Research Center is apparently caused by infection by a type D retrovirus. The complete nucleotide sequence (8173 base pairs) of a molecular clone of the prototype SAIDS virus isolate, SRV-1, reveals a typical retrovirus structure with long terminal repeats (346 base pairs) and open reading frames for the gag (663 codons), pol (867 codons), and env (605 codons) genes. SRV-1 also has a separate open reading frame of 314 codons between the gag and pol genes that defines the viral protease gene (prt) and a short open reading frame of unknown significance downstream from the env gene. The SRV-1 protease region shows a high degree of homology to its counterpart in the hamster intracisternal A-type particle genome; both these protease genes are about twice as long as the analogous region of other retroviruses. SRV-1 has no notable similarity in either genetic organization or sequence to the human AIDS retroviruses.     

杂交鲟和匙吻鲟HSP70 cDNA克隆与序列分析

采用RT-PCR法从杂交鲟(♀Huso huso×♂Acipenserschrencki)和匙吻鲟(Polyodonspathula)肝脏RNA克隆获得HSP70基因的全长cDNA(HSP70 cDNA)。所测定的HSP70 cDNA序列与NCBI/GenBank上登载的鲫(GenBank No.DQ872648)同源性最高,杂交鲟和匙吻鲟分别达96%和98%,杂交鲟HSP70序列为382 bp,匙吻鲟为334 bp。将杂交鲟和匙吻鲟与其它脊椎动物HSP70氨基酸序列用DNAstar软件进行相似度比较,鱼类与哺乳动物、两栖类非洲爪蟾之间HSP70氨基酸序列相似度平均值分别为86.2%和86.8%。鱼类之间的氨基酸序列相似度较大,平均为93.8%,表现出较高的保守性。以HSP70核苷酸序列为分子标记,用MEGA4软件中最大简约法(MP)构建了12个物种HSP70系统发育树,识别出3个大的单系类群:杂交鲟、匙吻鲟、团头鲂、鲫、鲤、斑马鱼聚为类群一(bootstrap 96);虹鳟和大西洋鲑聚为类群二(bootstrap 100);人类和褐家鼠类聚为类群三(bootstrap 89)。     

Primary structure and biochemical properties of an M2 muscarinic receptor

A partial amino acid sequence obtained for porcine atrial muscarinic acetylcholine receptor was used to isolate complementary DNA clones containing the complete receptor coding region. The deduced 466-amino acid polypeptide exhibits extensive structural and sequence homology with other receptors coupled to guanine nucleotide binding (G) proteins (for example, the beta-adrenergic receptor and rhodopsins); this similarity predicts a structure of seven membrane-spanning regions distinguished by the disposition of a large cytoplasmic domain. Stable transfection of the Chinese hamster ovary cell line with the atrial receptor complementary DNA leads to the binding of muscarinic antagonists in these cells with affinities characteristic of the M2 receptor subtype. The atrial muscarinic receptor is encoded by a unique gene consisting of a single coding exon and multiple, alternatively spliced 5' noncoding regions. The atrial receptor is distinct from the cerebral muscarinic receptor gene product, sharing only 38% overall amino acid homology and possessing a completely nonhomologous large cytoplasmic domain, suggesting a role for the latter region in differential effector coupling.