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。     

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]。     

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。     

红色角毛虫的形态学和形态发生过程的研究

观察并描述了上海采集到的红色角毛虫的形态结构和形态发生过程。发现形态发生时虫体分别于前、后两个区域发生前、后两个口围带原基,并且由同一个体分裂而成的前,后两个仔虫内,额、腹、横棘毛和缘棘毛的分化并不完全相同,以至造成两个仔虫上棘毛的数目和缘棘毛的列数有明显差异。根据红色角毛虫的形态结构及其形态发生中的一些不够稳定的特点,推测它可能是一种还处于分化中的腹毛类纤毛虫。     

伪尖毛虫Oxytricha fallax的形态学研究

伪尖毛虫Oxytricha fallax系原生动物门、纤毛虫纲、腹毛目、尖毛虫科的一种常见的单细胞动物。一般认为分裂间期的伪尖毛虫具有两个长圆形大核和两个圆球形小核。虫体腹面生有八根额棘毛、五根腹棘毛及五根肛棘毛,周缘左右两侧还生有缘棘毛,背面则有五列背触毛。 伪尖毛虫是原生动物中高度进化的类型,大、小核有显著的分化,细胞表面的纤毛小器官复杂而又明显,因此它是用以进行研究的一种良好材料。对它的核器与纤毛小器官以及它们在无性分裂过程中的发生和变化进行认真的探讨,可以使我们较详细地掌握该虫的形态特征,同时也能为进一步的研究提供一些资料。     

红色角毛虫生理改组过程的研究

红色角毛虫在生理改组时,随着老纤毛器的瓦解,先后出现新的口器,额、腹、横棘毛,左、右缘棘毛和背触毛四个原基区,并发生原基区的分化、新结构的形成和定位。这种新、老结构的更替过程相似于同种纤毛虫正常形态发生时期纤毛器的演化过程,口围带改组时,新口围带原基在左列中腹棘毛左侧的范围形成,后来,随着老口围带的瓦解,它向前方移动并处于老口围带的右侧,并继续朝老口围带位置移动、替换老口围带。这不同于其他常见的腹毛类纤毛虫,生理改组时新口围带原基在瓦解着的老口围带的位置逐渐移动替换老口围带的情况。     

阔口尖毛虫皮层纤毛器微管在不同生理条件下的分化

应用激光扫描共聚焦显微术,显示腹毛类纤毛虫阔口尖毛虫(Oxytricha platystoma)无性生殖过程中,新的口围带、波动膜、额腹横棘毛、左右缘棘毛微管先后分化,老纤毛器微管去分化,细胞分裂产生各含一套纤毛器微管的前、后两仔虫;生理改组过程中,口围带、波动膜、额腹横棘毛、左右缘棘毛微管发生去分化和再分化,细胞皮层微管胞器更新形成含一套纤毛器微管的新细胞。结果表明阔口尖毛虫在无性生殖和生理改组这两种不同的生理条件下,其纤毛器微管结构的形成或更新可能具有相同的细胞调控机制,形态发生中老纤毛器结构可能对新结构的发生和发育具有诱导定位和物质贡献的作用。     

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。     

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     

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.     

Nuclear transplant studies of the determination of mating type in germ nuclei of Paramecium tetraurelia

The micronucleus from vegetative cells of one mating type (O or E) in Paramecium tetraurelia was transplanted by micropipet into amicronucleate cells of opposite mating type (E or O). When autogamy was induced in the recipient cells, they developed new macronuclei and micronuclei derived from the transplanted micronucleus and usually expressed the same mating type as the recipients. The results indicate that micronuclei in the asexual phase may be undetermined for mating type. Recipient E cells in which the macronucleus had been previously removed were transplanted with a whole macronucleus from an O cell. Their mating type was soon transformed E to O before the occurrence of autogamy, and remained O after autogamy. This demonstrates that the transplanted macronucleus determined the O cytoplasmic state to determine the developing zygotic macronucleus for mating type O. It is unlikely that the micronucleus is determined for mating type in O or E cell during the asexual cycle.     

Inactivation of a macronuclear intra-S-phase checkpoint in Tetrahymena thermophila with caffeine affects the integrity of the micronuclear genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.     

Stomatogenesis during sexual and asexual reproduction in an amicronucleate strain of Paramecium caudatum.

Amicronucleate cells of Paramecium caudatum, whose micronuclei have been artifically removed by micropipetting, are characterized by the appearance of a deciliated area at the posterior part of the buccal opening. These cells form food vacuoles at a slightly lower rate than micronucleate cells. Their mean interfission time is longer than that in micronucleates. The exconjugants of amicronucleate cells can not form food vacuoles and eventually die witout fission, though conjugation proceeds normally in them as well as in their micronucleate mate. The oral apparatus of amicronucleate exconjugants seems to be shallower than that of micronucleates. The membranellar cilia, therefore, can be seen through the buccal overture by scanning electron microscope. The results obtained from the cross of micronucleate and amicronucleate strains and from the induction of autogamy in amicronucleate strains suggest that the micronucleus has a primary role in developing the normal oral apparatus after nuclear reorganization.     

Analysis of germinal aging in Paramecium caudatum by micronuclear transplantation

The role of the micronucleus in the age-dependent increase in mortality after conjugation in Paramecium has been investigated using micronuclear transplantation. The clone of Paramecium caudatum used for this study had a lifespan of about 750 fissions. In this clone, the fission rate began to decrease about 450 fissions after conjugation. Mortality after selfing conjugation also began to appear at about 450 fissions and gradually increased with clonal age. Cells at about 650 fissions showed 10–70% survival after selfing conjugation but when their micronuclei were transplanted into amicronucleate cells of about 450 fissions, the progeny survival increased to 70–90%. When micronuclei from cells 700–750 fissions old were transplanted into amicronucleate cells of 100–150 fissions, however, increase in progeny survival was very rare. The results indicate that micronuclei in cells up to the age of 650 fissions can function normally if the cytoplasmic environment is young.     

Immunofluorescence evidence for the absence of histone H1 in a mitotically dividing, genetically inactive nucleus

Antibodies directed against whole histone and purified lysine-rich histone H1 extracted from isolated macronuclei of the ciliate Tetrahymena were obtained and conjugated to fluorescein isothiocyanate. The fluorescein-antibody conjugates were used to directly label Tetrahymena cells. Both macro- and micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However, the anti-H1 conjugate only labeled macronuclei. This in situ demonstration of the lack of positive immunofluorescent staining of micronuclei with anti-H1 conjugate provide further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus.     

Genetic Analysis of Mating Type Differentiation in PARAMECIUM TETRAURELIA. II. Role of the Micronuclei in Mating-Type Determination

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.     

Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences.

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.