嗜热四膜虫SPO11基因缺陷型细胞株的基因表达变化分析

有性生殖的关键过程是通过减数分裂产生生殖细胞,而减数分裂的一个重要环节是进行基于DNA双链断裂的同源染色体重组。在同源染色体重组过程中,SPO11蛋白催化产生DNA双链断裂,从而起始同源染色体的重组。因此,研究SPO11基因缺失在减数分裂过程中所引起的基因表达变化有助于在转录组水平上了解该基因的作用。本研究通过对嗜热四膜虫(Tetrahymena thermophila)野生型和SPO11敲除细胞株在接合生殖时期2 h、3 h、4 h、5 h四个时间点的转录组进行高通量测序。通过差异表达基因分析和功能富集分析,发现SPO11基因敲除之后嗜热四膜虫在接合生殖时期2 h时,与DNA代谢过程和DNA复制相关基因的表达发生变化,推测SPO11基因敲除导致的减数分裂过程异常可能与DNA代谢过程和DNA复制相关。     

驱动蛋白Kin11调控嗜热四膜虫生殖系小核的减数分裂

驱动蛋白(kinesin)是分子马达蛋白质超家族成员,主要参与囊泡与细胞器的运输、纺锤体组装、有丝分裂和减数分裂等过程。在减数分裂期,不同驱动蛋白发挥功能的调控机制并不十分清楚。嗜热四膜虫(Tetrahymena thermophila)中含有14个驱动蛋白家族成员。其中,kinesin-6家族的唯一成员Kin11(TTHERM_00637750),在营养生长期低表达,饥饿期不表达,有性生殖期表达上调。Kin11编码1608个氨基酸,包含1个N端保守的马达蛋白结构域,C端卷曲螺旋(coiled-coil)结构域,并在N端和C端分别含有核定位信号NLS1和NLS2。Kin11在营养生长期和有性生殖期,定位在有丝分裂和减数分裂的小核和纺锤体上,并在有性生殖后期alignment阶段定位于小核上。Kin11与微管蛋白共定位于有丝分裂和减数分裂的纺锤体上。将Kin11的N端含有NLS1的1~400位氨基酸序列截短后,截断突变体定位在有性生殖减数分裂期的小核和纺锤体上。而将其C端含有NLS2的1008~1608位氨基酸残基截短后,截断突变体只能定位在有丝分裂和减数分裂后期的小核及有丝分裂的纺锤体上。敲除KIN11导致减数分裂过程中的纺锤体结构发生异常变化,小核染色体不均等分离与丢失,有性生殖发育停滞。结果表明,嗜热四膜虫驱动蛋白Kin11通过影响纺锤体结构,参与调控四膜虫生殖系小核在减数分裂过程中的正常分离。     

诱导嗜热四膜虫(Tetrahymena thermophila)细胞凋亡样变化的研究

本文以单细胞真核生物嗜热四膜虫(Te-trahymena thermophila)作为实验材料以抗肿瘤药物高三尖杉脂碱(Homoharringtonine,HHT)、糖皮质激素类药物地塞米松(9α-Fluo-ro-16α-methylprednisolone,Dex)和抗生素类药物放线菌素D(Actinomycin D)诱导嗜热四膜虫凋亡并研究其细胞凋亡过程的生物化学特性。结果表明抗肿瘤药物及抗生素类药物均不能明显地诱导嗜热四膜虫细胞凋亡。但糖皮质激素类药物在含一定量的Ca~(2 )、Mg~(2 )离子时能诱导嗜热四膜虫发生凋亡。作者认为诱导嗜热四膜虫凋亡过程可能与糖皮质激素类药物诱导鼠胸腺细胞凋亡的机制是类似的,嗜热四膜虫与胸腺细胞的凋亡过程可能同样被Ca~(2 )、Mg~(2 )离子依赖性的核酸内切酶的活化机制所控制着。     

四膜虫功能基因组数据库增量更新2016:生活史和减数分裂转录组及磷酸化蛋白组资源建设

原生动物嗜热四膜虫是一种优良单细胞真核模式生物,以其作为研究对象在基础生物学领域的研究已经取得了一系列突破性的成果。2006年,其大核基因组测序完成并发表,标志着四膜虫的研究进入了功能基因组时代。2013年基于基因芯片、基因网络和转录组数据,我们构建了四膜虫功能基因组数据库,其目前已成为模式生物嗜热四膜虫研究的两个重要数据库之一。在过去几年里,随着对四膜虫功能基因组学研究的深入,相关组学数据也实现了一定积累,对这些数据的整合也非常迫切。基于此,我们对四膜虫功能基因组数据库进行了增量更新。更新内容主要包括三个方面:(1)四膜虫生活史不同时期转录组数据;(2)四膜虫接合生殖减数分裂过程转录组数据;(3)磷酸化蛋白组数据。此次增量更新进一步提升和完善了四膜虫功能基因组数据库的内容和功能,对以四膜虫为对象的相关研究工作具有重要作用。     

金属离子促进嗜热四膜虫错配修复蛋白Mlh3核酸内切酶活性

嗜热四膜虫有性生殖过程中生殖系小核延伸并活跃转录,减数分裂过程中染色体同源重组起始于程序化的DNA双链断裂的形成,DNA错配修复系统能够去除DNA复制过程中所引起的错配并促进同源重组。减数分裂特异表达的错配修复因子Mlh3对四膜虫的有性生殖是必需的,然而具体功能并不清楚。本研究人工合成MLH3（TTHERM_001044369）基因,构建重组表达质粒pGEX-MLH3, 转化大肠杆菌BL21（DE3）并获得重组表达的GST-Mlh3蛋白。纯化的GST-Mlh3蛋白在配位不同的金属离子Cu2+、Mn2+、Mg2+后,有效切割超螺旋质粒DNA。ATP和ADP可进一步促进Mlh3的核酸内切酶活性。突变Mlh3中离子结合模体DQHA(X)2E(E)4E中的D117和E123位点,Mlh3D117N/E123A的核酸内切酶活性降低。进一步删除离子结合和ATP结合位点的C端结构域,突变体的核酸内切酶活性进一步降低,表明Mlh3的核酸内切酶活性是离子依赖型。减数分裂期HA-Mlh3免疫共沉淀鉴定了Mlh3可能的相互作用因子链交换蛋白Dmc1、DSB修复蛋白Mnd1、MutS、染色体维持蛋白Smc2和Smc4。结果表明,四膜虫的Mlh3通过金属离子依赖的内切酶活性,以及与其他因子相互作用,在减数分裂错配修复和同源重组过程中发挥重要作用。     

双氢青蒿素对嗜热四膜虫的毒性效应

以嗜热四膜虫(Tetrahymena thermophila)为试验对象, 双氢青蒿素(Dihydroartemisinin, DHA)以终浓度为0(对照组)、40、80、160和320 μmol/L 分别加入到嗜热四膜虫细胞培养液中, 探讨双氢青蒿素对嗜热四膜虫的毒性作用。采用 CCK-8 法检测嗜热四膜虫细胞增殖, 倒置显微镜和荧光显微镜观察细胞的形态结构及运动, 采用流式细胞术检测线粒体膜电位, 检测细胞内抗氧化还原酶活力和线粒体酶活力。结果表明, DHA显著抑制嗜热四膜虫增殖(P＜0.05), 在一定暴露时间内增殖活力和浓度呈负相关。双氢青蒿素作用嗜热四膜虫48h后各暴露组细胞皱缩变圆, 对照组细胞呈椭圆状。其中在160和320 μmol/L DHA暴露下, 嗜热四膜虫在培养基中的活动减弱, 细胞核出现固缩和浓染等特征, 线粒体膜电位显著下降(P＜0.05)。随着 DHA浓度增加, 细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和谷胱甘肽硫转移酶(GST)活性先增强后下降。线粒体内琥珀酸脱氢酶(SDH)活性逐渐降低, 与对照组相比, 差异均有统计学意义(P＜0.05)。上述结果表明, 双氢青蒿素对嗜热四膜虫具有毒性作用, 抗氧化酶在一定程度上能抵抗双氢青蒿素暴露导致的氧化损伤。氧化应激和线粒体损伤可能是双氢青蒿素对嗜热四膜虫产生毒性效应的重要机制。     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

过表达周期蛋白Cyc28影响四膜虫的有性生殖进程

真核生物的细胞周期通过连续的激活和失活特定的周期蛋白/周期蛋白依赖性激酶复合物活性进行调控。嗜热四膜虫含有34种周期蛋白,有性生殖期特异表达的周期蛋白Cyc2和Cyc17在四膜虫小核减数分裂中发挥重要功能。本研究从嗜热四膜虫中鉴定出一种新的周期蛋白CYC28 (TTHERM_00082190)基因,预测编码266个氨基酸。实时荧光定量PCR表明,CYC28在有性生殖时期特异表达,且在4 h表达水平最高。通过同源重组构建获得MTT1启动子调控下的HA-CYC28突变体细胞。免疫荧光定位表明,HA-Cyc28定位在细胞质和凋亡的亲本大核中。分别构建CYC28敲除突变株和RNA干扰细胞株,对CYC28敲减突变体细胞的分析发现,营养生长和有性生殖期突变细胞发育正常。然而,过表达株Cyc28突变体引起原核染色体排列异常,原核不能完成有丝分裂形成配子核,有性生殖进程终止。结果表明,Cyc28参与细胞的有性生殖进程,它的正常表达和降解对原核有丝分裂的完成是必需的。     

过表达周期蛋白Cyc28影响四膜虫的有性生殖进程北大核心CSCD

真核生物的细胞周期通过连续的激活和失活特定的周期蛋白/周期蛋白依赖性激酶复合物活性进行调控。嗜热四膜虫含有34种周期蛋白,有性生殖期特异表达的周期蛋白Cyc2和Cyc17在四膜虫小核减数分裂中发挥重要功能。本研究从嗜热四膜虫中鉴定出一种新的周期蛋白CYC28(TTHERM_00082190)基因,预测编码266个氨基酸。实时荧光定量PCR表明,CYC28在有性生殖时期特异表达,且在4 h表达水平最高。通过同源重组构建获得MTT1启动子调控下的HA-CYC28突变体细胞。免疫荧光定位表明,HA-Cyc28定位在细胞质和凋亡的亲本大核中。分别构建CYC28敲除突变株和RNA干扰细胞株,对CYC28敲减突变体细胞的分析发现,营养生长和有性生殖期突变细胞发育正常。然而,过表达株Cyc28突变体引起原核染色体排列异常,原核不能完成有丝分裂形成配子核,有性生殖进程终止。结果表明,Cyc28参与细胞的有性生殖进程,它的正常表达和降解对原核有丝分裂的完成是必需的。     

FISH分析栽培高梁×拟高梁、甜高梁×拟高梁的染色体行为

采用荧光原位杂交技术对45SrDNA在栽培高梁×拟高粱、甜高梁×拟高梁F,的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号,在减数分裂粗线期、终变期、中期Ⅰ染色体上45SrDNA位于一个二价体上,说明这两个杂种携带45SrDNA的染色体为同源染色体。根据45SrDNA位点随细胞减数分裂过程的位置变化,表明这两个杂种染色体配对行为正常,平均构型为2n=2x=20（10Ⅱ）,证明45SrDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

Germline and Somatic Transformation of Mating Tetrahymena Thermophila by Particle Bombardment

Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.     

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.     

药蒲公英减数分裂异常行为探讨

对药蒲公英减数分裂各期进行了观察,研究得出药蒲公英花蕾直径大小与花粉母细胞减数分裂各期之间的关系(花蕾直径在2-7mm时为减数分裂期)。并发现药蒲公英减数分裂中出现许多异常行为。如后期桥和落后染色体;药蒲公英花粉粒空瘪,这些异常行为的原因是减数分裂过程中有倒位和重复缺失等染色体结构变异出现以至形成双着丝点染色体。减数分裂过程的异常行为也说明药蒲公英是多倍体。     

The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus

Ciliated protozoans present several features of chromosome segregation that are unique among eukaryotes, including their maintenance of two nuclei: a germline micronucleus, which undergoes conventional mitosis and meiosis, and a somatic macronucleus that divides by an amitotic process. To study ciliate chromosome segregation, we have identified the centromeric histone gene in the Tetrahymena thermophila genome (CNA1). CNA1p specifically localizes to peripheral centromeres in the micronucleus but is absent in the macronucleus during vegetative growth. During meiotic prophase of the micronucleus, when chromosomes are stretched to twice the length of the cell, CNA1p is found localized in punctate spots throughout the length of the chromosomes. As conjugation proceeds, CNA1p appears initially diffuse, but quickly reverts to discrete dots in those nuclei destined to become micronuclei, whereas it remains diffuse and is gradually lost in developing macronuclei. In progeny of germline CNA1 knockouts, we see no defects in macronuclear division or viability of the progeny cells immediately following the knockout. However, within a few divisions, progeny show abnormal mitotic segregation of their micronucleus, with most cells eventually losing their micronucleus entirely. This study reveals a strong dependence of the germline micronucleus on centromeric histones for proper chromosome segregation.     

Unique sequences and predicted functions of myosins in Tetrahymena thermophila

Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.     

An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila.

Large palindromic DNAs are found in a wide variety of eukaryotic cells. In Tetrahymena thermophila, a large palindrome is formed from a single rRNA gene (rDNA) during nuclear differentiation. We present evidence that a key step in the formation of the rDNA palindrome of T. thermophila involves homologous intramolecular recombination. Heteroduplex micronuclear rDNA molecules were constructed in vitro and microinjected into developing macronuclei, where they formed palindromes. Analysis of the resulting palindromes indicated that both strands of the microinjected rDNA are used to form the same palindrome. This study, together with a previous study (L. F. Yasuda and M.-C. Yao, Cell 67:505-516, 1991), is the first to define a molecular pathway of palindrome formation. The process is initiated by chromosome breakage at sites flanking the micronuclear rDNA. An intramolecular recombination reaction, guided by a pair of short inverted repeats located at the 5' end of the excised rDNA, covalently joins the two strands of micronuclear rDNA in a giant hairpin molecule. Bidirectional DNA replication converts the giant hairpin molecule to a palindrome. We suggest that the general features of this pathway are applicable to palindrome formation in other cell types.     

Cytogenetic processes in the course of Triticum aestivum and Haynatricum hybridization

Meiosis in hybrids obtained in direct and return crossings between Haynatricum and wheat was studied. In F1 hybrids the possibility of stimulation of homoeologous pairing between the chromosomes of T. aestivum and T. dicoccum and, probably, H. villosum, if Haynatricum was used as pollinator, has been shown. This process is considerably intensified when the genes regulating chromosome pairing, in particular ph1b mutation, are used. In reciprocal crosses it was shown that wheat genotypes can differently influence on homoeologous chromosome pairing. In BC1 and F2 generations the chromosome compositions are determining which arise as a result of stochastic processes in premeiotic mitoses and in meiosis in F1 hybrids.     

Ingestion and inactivation of bacteriophages by Tetrahymena

Abiotic factors are thought to be primarily responsible for the loss of bacteriophages from the environment, but ingestion of phages by heterotrophs may also play a role in their elimination. Tetrahymena thermophila has been shown to ingest and inactivate bacteriophage T4 in co-incubation experiments. In this study, other Tetrahymena species were co-incubated with T4 with similar results. In addition, T. thermophila was shown to inactivate phages T5 and lambda in co-incubations. Several approaches, including direct visualization by electron microscopy, demonstrated that ingestion is required for T4 inactivation. Mucocysts were shown to have no role in the ingestion of T4. When (35)S-labeled T4 were fed to T. thermophila in a pulse-chase experiment, the degradation of two putative capsid proteins, gp23(*) and hoc, was observed. In addition, a polypeptide with the apparent molecular mass of 52 kDa was synthesized. This suggests that Tetrahymena can use phages as a minor nutrient source in the absence of bacteria.