筛选冠突伪尾柱虫有小核细胞系和无小核细胞系饥饿期差异表达的ESTs

利用显微切割的方法建立大型腹毛目纤毛虫---冠突伪尾柱虫(Pseudourostyla cristata)的无小核细胞系,分别提取有小核和无小核细胞系的总RNA,用SMART-PCR方法直接合成dscDNA,进而运用抑制性消减杂交技术(Sup-pression subtractive hybridization,SSH)对冠突伪尾柱虫去小核前后的差异表达基因进行研究,构建了有与无小核细胞系在饥饿期差异表达基因的消减文库,随机挑取了284个克隆,应用cDNA阵列技术鉴定阳性克隆。将鉴定出的17个阳性克隆进行Blastx分析,结果表明17个EST(Expressed sequence tag)均可能与小核体功能密切相关,此为进一步阐明小核的遗传机理奠定了基础。       

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     

念珠伪角毛虫小核体功能初步研究

为了研究念珠伪角毛虫的小核是否具有及具有怎样的体功能,采用显微切割手术去小核并建立无小核细胞系。经蛋白银染色鉴定,无小核细胞系群体中大多数细胞的形态结构存在缺陷：口围带的部分小膜缺损或排列紊乱,大核的数目和形态也不正常。这表明,念珠伪角毛虫的小核对于保持口围带结构的完整性以及大核的数目和形态结构的稳定性起着明确的作用。     

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。     

纤毛虫大核和小核的形态及其发育过程

纤毛虫细胞内同时含有大核(macronucleus)和小核(micronucleus)两种类型的细胞核。大核和小核在结构和功能等诸方面有明显差异。但一些进化程度较低的纤毛虫大核和小核没有进一步的分化,甚至极少数纤毛虫仅含一种类型的细胞核。本文就纤毛虫大、小核的形态及其发育过程的基本特征,以及某些低等纤毛虫核器的特殊情况概述如下。     

棘尾虫接合生殖期间核对形态发生的影响

本文完成了四项实验,即1.促成棘尾虫无小核系与有小核系接合;2.诱导无小核系自系接合;3.在正常接合对上摘除全部大核或一个接合体的大核及小核;4.在正常接合后体上对老大核碎块和新大核胚基进行损伤实验。以改进过的黑色素法显示这些实验的结果,发现第一次接合形态发生和小核的有性过程都主要是由大核支持的,小核在第一次形态发生中也起重要作用。这些核产物可从一个有核接合体穿过细胞质桥去支持另一无核接合体的发育。本文还发现接合第二次形态发生由新大核胚基控制。损伤染色体和多线染色体时期的大核胚基,第二次形态发生消失或不正带。越过第二次形态发生的大核胚基显著增强耐受损伤的能力,这些结果符合Prescott等(1973)对棘尾虫大核遗传装置的设想。     

包囊游仆虫包囊形成和解脱过程中大、小核的研究

包囊游仆虫形成包囊时大核除经历形态大小的变化外,大核DNA含量也低于正常大核水平;细胞脱包囊前,在大核一端或两端发生染色质粗浓集,是大核DNA复制的结果;染色质粒在整个大核内浓集时,大核DNA含量已达到正常大核水平,此时DNA复制结束,细胞脱包囊。小核在游仆虫形成包囊和脱包囊过程中,其形态大小、DNA含量等无明显变化。     

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     

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。     

双小核草履虫在遗传学研究中的应用

双小核草履虫(Paraecium aurelia)比尾草履虫(P.caudatum)较为小些(长约135微米,宽35—40微米),细胞内含有一个多倍体的大核和两个双倍体的小核(尾草履虫则含有一个大核和一个小核)。双小核草履虫的有性生殖及其遗传学双小核草履虫的接合生殖开始时,两个草履虫先在近前端的无纤毛区附着,以后,附着区逐渐扩大,咽道区的膜发生融合,两个接合体之间产生了细胞质通道。随着每个接合体内的大核渐趋瓦解,而所含的两个小核均发生减数分裂,经过连续二次分裂后,每个细胞内的小核数变为8个。接着每个细胞的8个小核有7个瓦解,剩下的各一个单倍体小核经有丝分裂产生两个配子核,其中一个是动核,另一个是静核。两个接合体中的动核经过细胞质通道各自移到对方细胞中,与对方的静核结合成合子核。两     

Reorganization in Amicronucleates with Defective Mouth of the Ciliate Pseudourostyla levis1

ABSTRACT. The purpose of this work is to examine the reorganization process in amicronucleates with defective mouth of the multimicronuclear ciliate, Pseudourostyla levis. The amicronucleates were derived from fragments obtained by transection of normal micronucleates. The cell size of the amicronucleates was extremely unstable and varied from 57.5 to 276.3 μm long (n = 146), whereas the micronucleates kept rather stable cell lengths with a range from 162.5 to 266.3 μm (mean ± SD = 213.1 ± 19.6 μm, n = 206). The renucleates obtained by transplantation of a micronucleus to an amicronucleate returned to almost normal cell size (mean ± SD = 203.7 ± 16.5 μm long, n = 54). Under the usual culture conditions in the amicronucleate cell line, the number of abnormal cells with defective mouth rapidly increased, up to about 60%, until a stationary phase. Similarly, abnormal cells also appeared in micronucleates although the frequency was always less than 10%. The mean number of membranelles in the normal adoral zone of membranelles (AZM) was 82.6 ± 3.9 (±SD, n = 49) vs. 57.3 ± 7.9 (n = 81) in ciliates with defective mouths. The missing part of the AZM was always the anterior part of the lapel. Cells with defective mouth underwent reorganization (= physiological regeneration) under the usual culture conditions. During reorganization, the lapel part of the old AZM was transformed into a new collar part. The defective mouth was repaired through this developmental process. These results suggest that in P. levis the decrease of food supply often leads to the loss of a specific part of the AZM and that this membranellar loss is suppressed by the existence of micronuclei.     

冠突伪尾柱虫（Pseudourostyla cristata）实验再生研究

冠突伪尾柱虫(Pseudvurostyla cristata) 含约70枚大核。我们用显微手术横切G1期细胞,得前后两块相等断片;分别培养。60小时后,断片再生完成。在再生过程中,随细胞体积增大,大核数目也增加。大核的数目和细胞体积存在着一定的均衡关系。在细胞无性分裂过程中,许多大核改组后,融合成一个融合大核。这个融合大核具两个仔虫的大核数目和DNA量。我们用显微手术得到含融合大核的后断片。在后断片再生后恢复的虫体内,我们发现本应分配到两个仔虫中去的大核数目,被限制在一个虫体的大核数目上。这说明了细胞质可以影响和调节大核的数目。并还证明了这种虫体大核DNA量较正常虫的大核DNA量约多一倍。其中大部分虫体分裂时,大核不经改组就开始融合和分裂;从而使DNA量回复正常。同讨还发现小部分虫体通过排出大核多余核物质方式来调节大核DNA量。这些现象说明了细胞核质之间存在着一种调节相对平衡和相互协调的机制。     

沼泽瘦尾虫皮层纤毛器及其附属微管的观察

应用扫描电镜技术、荧光紫杉醇直接荧光标记显示了腹毛目纤毛虫沼泽瘦尾虫(Uroleptus limnetis)的细胞形态和皮层纤毛器的组成模式,以及皮层口围带、额腹横棘毛、左右缘棘毛等纤毛器微管和纤毛器附属微管的建构特征,可为进一步阐明瘦尾虫类纤毛虫的形态学及其系统发育研究提供基础资料。     

棘尾虫对折右纵断片的再生和纤毛模式形成的研究

应用切割嫁接方法获得的棘尾虫完全对折和部分对拆右纵断片都经历了一面调整极性、一面再生和形态发生的过程。推测这两种断片在纤毛模式形成中发生的诸多现象可能与所在断片再生时的极性调整变化状态有关。     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

一种游仆虫无性分裂生殖的研究Ⅱ.无性分裂过程中皮层结构的形态发生

应用光学显微镜和扫描电子显微镜,观察到在一种游仆虫无性生殖周期中,新口围带发育时老口围带的更新、新波动膜原基的发生、棘毛原基发生的最早形态和背触毛发生等在其他种游仆虫中未见报道的现象。