棘尾虫接合生殖期间大核功能的研究

为了澄清棘尾虫接合期间大核对小核发育和皮层形态发生的作用,完成了大核摘除,放线菌素D处理,H~3-尿嘧啶核苷标记等实验。摘核实验证明,一个接合体的大核可以通过细胞质桥支持另一除去大核的接合体发育。即使保留接合对大核总数的1/8,这种支持作用仍然存在。还发现来自大核的支持物作用于形态发生更易于作用于小核发育,并对两个接合体的形态发生作用相等,对两个接合体小核发育的作用不等。摘除四分之三大核的实验证明,小核发育和皮层更新在接合后15小时内都不能脱离对残留大核的依赖。放线菌素D处理实验证明,接合后RNA的合成需积累到8.5小时,才可满足核与皮层发育的需要。H~3-尿嘧啶核苷标记实验也支持接合后前9小时内RNA都在持续合成的结论。本文对摘核实验和放线菌素D处理实验结果的差别、以及本文结果与前人结果的差别都做了讨论。     

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。     

第四双小核草履虫减数分裂产物的退化特征分析

通过吖啶橙和Hoechst 33342两种活体荧光染料双染的方法对第四双小核草履虫(Paramecium tetraurelia)接合生殖过程中小核减数分裂产物进行观察,结果发现位于口旁锥外的小核分裂产物呈蓝绿色或黄绿色,表明它们以凋亡的方式发生退化.     

上海四膜虫的胞质配合

对上海四膜虫（Ｔｅｔｒａｈｙｍｅｎａ ｓｈａｎｇｈａｉｅｎｓｉｓ）有小核细胞和无小核细胞的接合过程进行了跟踪观察,发现在四膜虫中也存在胞质配合（ｃｙｔｏｇａｍｙ）。在接合对中,有小核细胞一方可以完成正常的减数分裂过程,既有配子核的形成、配子核的融合、配子核分裂,最终形成两个有小核的子细胞。虽然有小核细胞的配子核并不进入无小核细胞,但是两细胞间却有细胞的迁移与交换。在整个接合过程中,无小核细胞的大核     

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     

核rDNA的ITS序列在被子植物系统与进化研究中的应用

被子植物与其它高等真核生物相似,核rDNA是高度重复的串联序列。由于同步进化的力量,绝 大多数物种中这些重复单位间已发生纯合或接近纯合。核rDNA的内转录间隔区(ITS)包含被5.8S rDNA所分隔的ITS1和ITS2两个片段,ITSl的长度为187～298bp,ITS2为187～252bp,经PCR扩 增后可以方便地对这两个片段进行直接测序或克隆测序。ITS序列变异较快,可以提供较丰富的变异 位点和信息位点,已证实它是研究许多被子植物类群系统与进化的重要分子标记,不仅可用于解决科、 亚科、族、属、组内的系统发育和分类问题,而且可用于重建多倍体复合体的网状进化关系,探讨异源多倍体的起源过程,然而,正是由于ITS序列变异较快,它一般不适于科以上水平的系统发育研究。     

棘尾虫有性生殖的发动和诱导自配的研究

本文观察了棘尾虫的接合反应。按照接合早期接合状态的变化,将接合反应分成10个时期,并将不同时期的接合对人工拆开,令其继续发育,然后计算每一时期中由营养虫转变为有性过程的虫体的百分比。结果表明,有性过程的发动是在Ⅳ期。一旦发动即不能逆转。将Ⅳ期被拆开的虫体横切成相等的前后切块,发现一些前切块由营养状态转变为有性状态,发生了自配,而所有后切块保持营养状态,发生了再生。这一现象意味着有性过程的发动不是整个细胞的一种突然改变,看来是一种从前向后的信息传递过程。用上述人工方法诱导出棘尾虫的自配,核器发生的观察表明,自配体中的桉行动和自配后体中核胚基的发育,所有这些核的事件都与接合生殖的相似,只不过没有两个配偶之间迁移原核交换罢了。对伴随着自配发生皮层重组也做了观察,自配的形态发生也与接合中的一样。     

核基因序列在昆虫分子系统学上的应用

核基因中含有更加丰富的生物学信息,运用核基因序列或将核基因序列与线粒体基因序列相结合研究昆虫的系统发育正成为分子系统学领域的一种发展趋势.核糖体基因中18S rDNA、28S rDNA、ITS已在昆虫分子系统学中得到了广泛的应用.与核糖体基因相比,虽然编码蛋白的核基因应用于昆虫分子系统学的种类不少,但大部分都是应用于双翅目和鳞翅目昆虫的分子系统学研究中,能够成功地普遍用于多个目昆虫的系统学研究的核基因并不多.本文简要介绍了应用于昆虫分子系统学的核中核糖体基因和编码蛋白的核基因,并分析了核基因序列在分子系统学应用上的局限性和应用前景.     

不同染色方法揭示纤毛原生动物草履虫接合生殖过程的新细节

通过石炭酸品红、Hoechst 33342、蛋白银及免疫荧光标记等染色方法对草履虫接合生殖过程进行了重新观察,结果发现:1)新月核是第一次减数分裂前期小核的主要形态学特征,在核内有一未被石炭酸品红、Hoechst 33342着色区域,蛋白银染色则清楚显示该结构;2)4个单倍体减数分裂产物中的1个核进入口旁锥完成配前第三次核分裂,其余3核退化.蛋白银染色和抗α微管蛋白单克隆抗体进行免疫荧光标记显示,核进入口旁锥的时期在第二次减数分裂末期而非减数分裂结束后;3)配前第三次分裂末期,核间连丝的中间段有一被蛋白银识别的结构,但免疫荧光标记却无显示,只表现为纤维状结构与两侧核间连丝相连.观察结果为草履虫接合生殖过程中相关分子生物学机制研究奠定了必要的形态学基础.     

冠突伪尾柱虫(Pseudourostyla cristata)接合生殖的研究 Ⅰ.核器演化

在25℃条件下,冠突伪尾柱虫接合生殖全程历时10天左右。接合生殖过程中的核器演化包括:①数十枚老的大核逐步瓦解。电镜观察表明,老的大核是以一种类似于食物泡消化的方式被吸收的,并在此过程中伴有大量溶酶体出现。②仅8枚左右小核中的一枚参与新核器的发生。首先,位于胞口后部的一枚小核膨大并进行一次预备分裂,接着发生三次成熟分裂。每一接合体内形成一枚雄原核和一枚雌原核。雄原核互向对方迁移并与其雌原核融合成为合子核。合子核分裂两次,四枚子核之一发育为大核原基,另一枚发育为小核原基,其余两枚退化。预备分裂和前两次成熟分裂各自产生的两枚子核中,仅一枚进入下一次分裂,另一枚解体消失。在第一次成熟分裂前期,“降落伞”的形成和发展经历着复杂的结构变化,持续一小时以上。③大核原基经过长时间的发育,伴有多线染色体的形成和解体等一系列变化,方达成熟状态。成熟的大核原基以伸长断裂、分叉断裂和哑铃形缢缩三种方式进行分裂,小核原基亦随之分裂,逐步形成具60枚左右大核、8枚左右小核的正常营养体。其后,大核融合,开始配后第一次无性分裂。值得注意的是,大核原基发育到将成熟时,最初的迹象是染色质向大核原基中央集结成团,染色质团与核膜之间充满着匀质的核液。当中央染色质团伸长时,又将     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila,as detected by in situ hybridization

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with ³H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei had disappeared. The hybridization in the anlage then decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post-conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non-irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.     

Conjugation-Specific Genes in the Ciliate Euplotes crassus: Gene Expression from the Old Macronucleus

ABSTRACT. Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene ( conZ47 ) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZ47 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.