大型纤毛虫总RNA的提取方法

以冠突伪尾柱虫为材料,建立了一种适合于富含RNase和多糖的大型纤毛虫总RNA的提取方法。该方法采用异硫氰酸弧和β-巯基乙醇联合快速变性,酚、氯仿和异戊醇抽提,同时在变性剂存在的条件下两次选择性地沉淀RNA,从而有效地去除了DNA、蛋白质和多糖。所得RNA样品经电泳、紫外分光光度法检测和RT-PCR分析,证实为完整、均一且无基因组污染的总RNA.这为构建冠突伪尾柱虫有小核系与无小核系之间的削减文库、筛选两细胞系差异表达的基因奠定了基础。     

瘦尾虫的小核在无性繁殖周期中对细胞形态结构的影响

通过显微切割建立瘦尾虫无小核细胞系,并与原细胞系及切割后再生的有小核细胞系进行对照观察。结果表明,无小核细胞的形态结构,尤其是口器出现高比率的畸形。在生长静止期,无小核细胞系群体中约23 % (36/155)的细胞完全失去了波动膜。与此同时,这些细胞的口围带也显出异常。一些无小核细胞的大核也出现异常,有的细胞仅含有1枚大核,有的则含有4枚,而不是通常的2枚;还有少数细胞的大核在非细胞分裂期进行分裂。上述结果提示,在无性繁殖周期中,瘦尾虫的小核对于维持正常的细胞形态结构、尤其是保持胞口结构的稳定性和大核数的恒定起着重要的作用[动物学报52 (2) : 383 -388 , 2006]。     

冠突伪尾柱虫有性生殖期间皮膜发育的核控制

通过显微手术去小核建立多个冠突伪尾柱虫（Pseudourostyla cristata)无小细胞系,并诱导它们与有小核细胞进行接合生殖,以评估小核及其衍生的大核原基在有性生殖期间对皮膜形态发生的影响,当无小核接合体从有小核配偶获得1枚配子核后,接合双方不仅能平行地继续核器演化,而且使第1次皮膜改组能够同步进行和正常发育,说明小核在有性周期中除了生殖功能外仍保留着某些控制皮膜发育的体功能,虽然大部分接合后体的大核原基在DNA贫乏期停止发育,但少数接合后体能够超越这一时期,并启动第2次皮膜改组和顺利完成其后续的有性发育全程,表明指令发动第2次皮膜发育的信号来自DNA贫乏期后以排出一核物质团块为标志的大核原基。     

冠突伪尾柱虫接合过程中小核的形态发生作用

为查明有性生殖期间小核对形态发生的影响程度和范围 ,我们利用蛋白银染色对冠突伪尾柱虫不同交配型无小核体之间的接合过程进行了跟踪观察。结果表明 :无小核细胞的交配反应能力明显下降 ;接合双方第一次皮膜形态发生如常进行 ,但纤毛器原基分化往往出现异常 ;接合后体照常进入拟包囊阶段 ,但原有纤毛器多不从皮膜上消失 ;由于没有新核器形成 ,均不能启动第二次形态发生并于拟包囊早期解体死亡。因此 ,小核对于维持接合早期及第一次皮膜改组的正常进行具有某些明显作用 ,其衍生的大核原基对于启动后续的发育则是绝对必要的     

冠突伪尾柱虫小核对胞口结构稳定性的影响

在通过显微手术所建立的冠突伪尾柱虫（Pseudourostyla cristata)无小核系中,部分细胞因口围带翻领部小膜出现不同程度的缺损而导致畸形;在细胞生长静止期的群体中,口围带畸形率高达50％以上;对无小核系600多个刚完成二分裂的前,后仔虫及1000多个生理改组末期的虫体进行蛋白银染色观察,除发现4个二分裂仔虫和3个生理改组后虫体的口围带异常外,绝大多数经过形态发生后的细胞都具有正常的胞口结构。另一方面,选择性培养实验进一步证明,口围带畸形产生于非形态发生期。由此可见,失去小核的细胞,同时也失去了胞口结构的稳定性。     

棘尾虫无小核镜像骈体的获得及其生殖行为的实验观察

用在分裂期进行显微手术的方法和在陈旧培养中诱导的方法,从贻贝棘尾虫天然无小核系S_(10)中获得了无小核镜像骈体。对骈体的各种生殖行为进行了观察,并用孚尔根和改进的黑色素染色技术揭示了它们的细胞学细节。发现在本文描述的无小核骈体和过去报告的有小核骈体之间,在二分裂和生理再生上没有显著区别。无小核骈体和正常单体S_7之间的接合命运则是多样化的。在630个这样的接合对中,有257对发生了真正的接合并形成接合后体;有153对接合不久包囊化;在其余的220对中,一个接合体吸收了另一个,变成营养型的单体和有小核的骈体。前者占总吸收者的96.8％,后者只占3.2％。对接合和吸收两种情况的细胞学事件进行了详细观察。无小核骈体中的两组细胞质和双套大核能吸引其配偶中的小核,形成自己新的双套大核胚基及小核胚基,在本文的讨论中受到充分注意。     

长江口地区五种腹毛类纤毛虫的形态学研究

利用活体观察和蛋白银染色技术, 对采自长江口泥滩潮间带及河道的五种腹毛类纤毛虫进行了形态学研究。包括对两个国内新记录种仁川巴库虫Bakuella (Bakuella) incheonensis和希斯多利织毛虫Histriculus histrio提供了详细的活体特征和纤毛图式信息, 对红色伪角毛虫Pseudokeronopsis rubra、黄色伪角毛虫Pseudokeronopsis flava和冠突伪尾柱虫Pseudourostyla cristata进行了形态学重描述。五种腹毛类纤毛虫的长江口种群与国内外种群均存在不同程度的形态学差异: 仁川巴库虫较韩国原始种群体型大, 口围带小膜和额前棘毛数较多; 希斯多利织毛虫与部分国外种群存在体长差异; 红色伪角毛虫与青岛种群相比形态特征基本吻合但个体大小的波动范围较大; 黄色伪角毛虫与湛江种群相比额棘毛数目较多; 冠突伪尾柱虫较奥地利种群体型较小, 与日本种群相比大核较多。该工作丰富了中国腹毛类纤毛虫多样性的认识。     

多节核棘尾虫无小核系虫体的初步研究

纤毛虫细胞具有大核和小核之分。多年来认为大核控制当代无性系细胞的全部代谢活动,小核只在有性生殖时起产生两性原核和受精的作用,从而更新无性系。近年来的研究证明,小核对当代无性系细胞的皮层形态及形态发生也起相当重要的作用。这此工作是在草履虫上(Ng等,1981,1984,1987)和贻贝棘尾虫上(史新柏等,1983,1990;Lu等,1989)完成的。但小核对大核的影响在任何纤毛虫上尚未见报道。笔者用所获得的新种多节核棘尾虫     

冠窦伪尾柱虫接合过程中小核的形态发生作用

为查明有性生殖期间小核对形态发生的影响程度和范围,我们利用蛋白银染色对冠突伪尾柱虫不同交配型无小核体之间 的接合过程进行了跟踪观察。结果表明：无小核细胞的交配反应能力明显下降;接合双方第一次皮膜形态发生如常进行,但纤毛器原基分化往往出现异常;接合后体照常进入拟包囊阶段,但原有纤毛器多不从皮膜上消失。由于没有新核器形成,均不能启动第二次形态发生并于拟包囊早期解体死亡。因此,小核对于纤维接合早期及第一次皮膜改组的正常进行具有某些明显作用,其衍生的大核原基对于启动后续的发育则是绝对必要的。     

伪狂犬病病毒gC基因在IBRS-2细胞中的表达及其对病毒繁殖的影响

 对含伪狂犬病病毒 Ea株 g C全基因的质粒 p UC1 .75进行亚克隆 ,将其完整编码区置于真核表达载体 pc DNA3.1 +的 HCMV启动子 /增强子下游 ,构建了 g C基因真核表达质粒 pc DNA-g C.脂质体转染 IBRS- 2细胞 ,在 G41 8抗性选择下 ,获得多个阳性克隆细胞系 .经 ELISA筛选反应最强的阳性克隆细胞系 ,进一步用间接免疫荧光检测证实 g C基因在 IBRS- 2细胞中得到了正确表达并分布在细胞膜 .以 1 0 0个蚀斑形成单位的伪狂犬病病毒分别接种表达 g C的细胞系和空白载体转染细胞系 .通过测定蚀斑数发现 ,表达 g C的细胞系对病毒的感染具有抑制作用 ,平均抑制率达 59.4%± 1 .3% .     

Evidence for Micronuclear Function During Vegetative Growth and Reproduction of the Ciliate, Tetrahymena pyrijormis

An amicronucleate clone of Tetrahymena pyrijormis has been found among the asexual progeny of irradiated cells of strain EU 6000 (variety 6, mating type I). Log-phase cells of this clone, designated EU 6525, have a mean generation time (6.0 hr) longer than that of the micronucleate strain, EU 6000 (2.9 hr). Further irradiation studies of strain EU 6000 indicate that the recovery of viable amicronucleate populations is rare although many amicronucleate cells are found among surviving progeny.¹ Attempts to introduce micronuclei into amicronucleate cells of strain EU 6525 by conjugation have been made. Micronucleate lines are obtained from amicronu create pair members only in low frequency. These results, considered together with those of other workers, suggest that some change in the state of the cell, additional to the physical loss (or gain) of the micronucleus, must occur before viable amicronucleate clones can be obtained from micronucleate cells, or before amicronucleate cells can produce viable micronucleate lineages. An alteration in mean generation time may be a reflection of this change, or it may simply be a direct consequence of micronuclear removal. The results further imply that the ciliate micronucleus unquestionably contributes information to the cell during asexual growth and reproduction.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Isolation of a single messenger RNA and of the corresponding gene in Plasmodium berghei

A genomic library of Plasmodium berghei DNA was constructed using lambda 47.I as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of the library using labelled cDNA complementary to the polyA + Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cβ3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13. Received: 22 July 1998 / Accepted: 24 August 1998     

小鼠Uncv基因克隆及真核表达

目的 克隆小鼠的Uncv基因并在真核细胞表达.方法 采用RT-PCR方法扩增小鼠皮肤组织中Uncv基因编码区,以真核表达质粒pcDNA 3.1-Flag为载体,构建Uncv真核表达质粒,将重组载体转染Hela细胞并用Western blot法检测基因表达.结果 构建Uncv基因真核表达载体pcDNA 3.1-Flag/Unev,重组质粒在Hela细胞中有效表达约95×103的融合蛋白.结论 成功构建真核表达载体pcDNA 3.1-Flag/Uncv,并且在真核细胞中有效表达,为研究Uncv基因生物学功能奠定基础.     

Nucleotide sequences and novel structural features of human and Chinese hamster hsp60 (chaperonin) gene families

A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.