依赖雄激素的大鼠储精囊分泌蛋白的离子交换层析纯化

本文报道利用阳离子交换层析,纯化了雄激素依赖的大鼠储精囊分泌蛋白(SVPⅡ、SVPⅣ、SVPⅤ_a、SVPⅤ_b及SVPⅥ)。主要纯化步骤包括下列二步:1.Sep-hadex G-100凝胶过滤;2.上样后,联合使用盐梯度和pH梯度,洗脱快速蛋白液相层析(FPLC)系统的阳离子交换柱Mono S。洗脱峰的纯度以变性条件下的聚丙烯酰胶凝胶电泳(SDS-PAGE)和等电聚焦(IEF)鉴定;借此,还测定了已纯化的大鼠储精囊分泌蛋白的分子量和等电点。     

大鼠前列腺上皮细胞角蛋白8mRNA表达调节的定位研究

本文应用反义RNA探针原位杂交法,研究雄激素对大鼠腹侧前列腺(VP)上皮细胞角蛋白(CK)8 mRNA表达的影响。发现1.在任何VP组织切片中,CK 8探针专一、大量定位于VP腺上皮细胞中,CK 8 mRNA是前列腺上皮细胞特异而灵敏的标志。2.去睾大鼠VP CK 8 mRNA染色增强,提示CK 8mRNA有过度表达,注射雄激素又可抑制其过度表达。3.与已知受雄激素抑制性基因不同,即使大鼠VP完全萎缩之后达2个月之久,其存留腺上皮细胞CK 8 mRNA表达仍持续增高。4.前列腺发育早期,迅速增殖的幼稚腺上皮细胞高度表达CK 8 mRNA,以后随着体内雄激素水平升高,VP上皮CK 8 mRNA表达下降,分布转移。以上结果进一步支持前列腺CK 8基因是新的一类受雄激素抑制性基因的推测,同时表明前列腺CK 8基因的表达与前列腺干细胞的增殖分化有密切联系,CK 8 mRNA高度表达是前列腺干细胞一个重要特征。     

大鼠前列腺素D合成酶在毕赤酵母中的表达及其在精子中的分布

研究大鼠L PGDS在毕氏酵母中的表达 ,探讨L PGDS在大鼠精子中的分布。以大鼠睾丸mRNA为模板 ,经逆转录获得L PGDS全长和成熟基因片段。将该基因片段克隆到pPIC9载体上 ,将重组表达质粒转化巴斯德毕赤酵母 (Pichiapastoris)GS115 ,筛选mut⁺表型 ,经甲醇诱导实现L PGDS的分泌表达。用SDS-PAGE分析L-PGDS的分子量 ,用PAS反应鉴定糖链 ,用免疫组织化学法研究L PGDS的分布 ,用Westernblot分析重组L-PGDS与精浆中L-PGDS。筛选出 4株表达水平较高的酵母工程菌株 ,SDS-PAGE分析表明 ,产物分子量约为27kD ;L-PGDS主要分布在精子的头部以及尾部的上段     

大鼠白蛋白mRNA3''''端顺序的分子克隆(简报)

白蛋白与甲胎蛋白基因是由同一祖先基因进化而来。在小鼠,它们是一前一后定位在同一条染色体DNA上。在个体发育和肝癌变过程中,这两基因的相互关系也呈现相反的表达水平。因此,研究甲胎蛋白基因表达的调控与白蛋白基因是不可分割的。本文简要报道我们克隆了大鼠白蛋白mRNA 3′端顺序,为研究白蛋白基因结构和表达提供了必要的探针。白蛋白mRNA是从Wistar大鼠肝脏用双抗体免疫沉淀法提取。分子克隆技术基本是按“分子克隆”实验手册进行。     

雄激素受体在大鼠睾丸中转录模式的分析

目的：研究Ar（雄激素受体）基因在大鼠睾丸组织中的转录模式。方法：取刚出生的雄性Wistar大鼠,于出生2~65日的不同时间点,脊椎脱臼处死3只不同窝别的幼鼠,提取睾丸组织总mRNA;将mRNA反转录成cDNA,随后采用Real-time PCR方法检测大鼠睾丸组织中Ar mRNA的转录情况。结果：Ar mRNA表达在出生后第2天开始缓慢上升到第16天达到最高值,第16天到第30天迅速下降,第31天出现一个小高峰后到第65天持续缓慢下降。结论：Ar基因在大鼠早期发育睾丸组织中表达量逐渐升高,可能与精原干细胞的增殖及初级精母细胞的发生相关。     

人骨形态发生蛋白2(BMP-2)在巴斯德毕赤酵母中的表达

以重组质粒pUC-BMP2为模板PCR扩增人BMP-2成熟肽编码序列,将该序列克隆入pGEM-T载体进行DNA序列分析后亚克隆入分泌型毕赤酵母表达载体pPIC9K中。重组质粒oPIC9K-BMP2经BelⅡ酶切后回收线性化片段,聚乙二醇法转染毕赤酵母茵株GS115。PCR筛选整合有人BMP-2基因的酵母细胞重组子,以甲醇进行诱导表达,于酵母细胞培养基中可检测到rhBMP-2。体外培养条件下,所得rhBMP-2可增加2T3小鼠成骨细胞内碱性磷酸酶活性;体内实验．rhBMP-2冻干粉可于小鼠骨四头肌肌袋中诱导软骨细胞群产生。     

致犊牛脑膜炎大肠杆菌新疆分离株ibeB基因的特性分析北大核心CSCD

【目的】分析致犊牛脑膜炎大肠杆菌分离株ibeB基因的分子生物学信息。【方法】以自脑炎死亡犊牛脑组织、肝组织分离鉴定的O161-K99-STa致病性大肠杆菌牛-EN株和牛-EG分离株为材料。根据GenBank中公布的脑膜炎大肠杆菌K1株RS218 ibeB基因序列设计1对引物,采用PCR方法,从分离株中成功克隆ibe B基因,比较分离株ibeB基因与不同来源大肠杆菌ibeB基因的部分生物信息学特性。【结果】分离株ibeB基因序列全长1500 bp,包含1371 bp开放阅读框,共编码457个氨基酸;生物信息学分析显示,牛-EN株与致人脑膜炎大肠杆菌K1 RS218的核苷酸和氨基酸同源性分别为90.5%和96.9%,牛-EG株与大肠杆菌K12的核苷酸和氨基酸同源性分别为99.4%和100.0%;ibeB蛋白为亲水性蛋白,分子质量为50.26 kDa,理论等电点为6.05;该蛋白无跨膜区,但具有信号肽序列;亚细胞定位显示,分泌信号通路位点(SP)占比例为0.939,说明该蛋白属于分泌型蛋白。【结论】从致脑膜炎大肠杆菌分离株中成功克隆ibeB基因,该基因与致人脑膜炎大肠杆菌K1 RS218 ibeB基因有较高的同源性,均有相似的生物学特性,属肠外致病性大肠杆菌。     

乙型肝炎病毒全长PreS蛋白基因在酵母中的表达

应用直接提取法从武汉地区乙型肝炎病人患者阳性血清中提取HBV基因组,以此作为模板,用引物PCR方法获得全长preS基因,该片段的DNA大小1203bp-克隆到pUCm-T载体中,应用M13通用引物进行序列测定,与中国HBV标准株序列比较,证实基因型为Adr亚型。有24个核苷酸不同,preS基因包含3个起始密码ATG分别为preS1,preS2,和S的翻译起点。然后将preS基因克隆到毕赤酵母表达载体pPIC9K中,将Sal Ⅰ线性化的pPIC9K—preS质粒用电击法导入巴斯德一毕赤酵母GS115His中。通过MD—G418平板筛选和5‘‘‘‘‘‘‘‘AOXI和3’AOXI引物PCR鉴定获得稳定重组HBV全长严preS基因的His^ Mut^ 酵母工程菌株。此酵母菌株在合适的培养条件和甲醇诱导下高效表达产生全长preS蛋白并可分泌到培养液中,SDS—PAGE显示培养液中含有分子量约为48kDa的带;微孔ELISIA法证实表达并分泌到培养液中的preS蛋白能够与HBV抗体阳性血清发生特异性反应。     

雄激素与大鼠前列腺细胞基因的表达

本文讨论了雄激素与大鼠前列腺细胞基因表达的关系。利用cDNA克隆和杂交等技术,已发现前列腺中有近十种依赖雄激素的蛋白质,本文较详细讨论了雄激素时甾体激素结合蛋白(PBP)及20K蛋白质基因表达的影响。激素受体复合物在核内主要结合在染色质上,这种结合导致染色质结构活化和专一性的基因表达。雄激素还可通过引起Poly(A)聚合酶活性增加等变化影响转录后加工。最后讨论了靶组织染色质结构特点及染色质上受体复合物结合位点的分布。     

APOBEC3F的克隆、真核表达及其抗HBV效应的初步研究

目的克隆、体外真核表达载脂蛋白BmRNA编辑酶催化多肽样3F（APOBEC3F）基因并研究其抗病毒效应。方法应用RT—PCR的方法从人PBMC细胞中扩增APOBEC3F基因,构建HA—APOBEC3F融合蛋白真核表达载体pXFA3F,pXFA3F和具有复制能力的1．3倍HBV载体pHBVl．3共转染HepG2细胞,Western印迹检测APOBEC3F融合蛋白在HepG2细胞中的表达,ELISA方法检测细胞培养上清液中的HBsAg和HBeAg水平,实时定量PCR检测HBV相关mRNA水平变化。结果克隆了APOBEC3F基因,其经测序与GeneBank公布序列一致;构建的APOBEC3F真核表达载体pXFA3F经转染可在HepG2细胞中瞬时表达;APOBEC3F可抑制乙型肝炎表面抗原和e抗原的分泌,可使转染细胞内HBV相关mRNA水平下降。结论成功克隆并真核表达了APOBEC3F基因,其在体外具有抗HBV生物学效应。     

Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.     

Molecular cloning of pea mRNAs encoding a shoot-specific polypeptide and light-induced polypeptides

Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of M_r27 500 and isoelectric point of 4.7.     

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

The expression of a plant genome in hnRNA and mRNA.

Representation of genomic kinetic sequence classes and sequence complexities were investigated in nuclear and polysomal RNA of the higher plant Petroselinum sativum (parsley). Two different methods indicated that most if not all polysomal poly(A) -RNA is transcribed from unique sequences. As measured by saturation hybridization in root callus and young leaves 8.7% and 6.2%, respectively, of unique DNA were transcribed in mRNA corresponding to 13.700 and 10.000 average sized genes. Unique nuclear DNA hybridized with an excess of polysomal poly(A)mRNA to the same extent as with total polysomal RNA. 3H-cDNA - poly(A)mRNA hybridization kinetics revealed the presence of two abundance classes with 9.200 and about 30 different mRNAs in leaves and two abundance classes with 10.500 and 960 different mRNAs in callus cells. The existence of plant poly(A)hnRNA was proven both by its fast kinetics of appearance, its length distribution larger than mRNA, and its sequence complexity a few times that of polysomal RNA.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.