组蛋白伴侣在发育过程中的功能

组蛋白伴侣能够协助组蛋白参与染色质的解凝和组装, 从而调控基因的表达, 对动植物的配子发生、受精、胚胎发育以及生长、衰老等发育过程都具有重要作用。文章主要对目前研究较多的组蛋白伴侣 - 核质蛋白、CAF-1、HIRA、ASF1/CIA及NAP1在发育过程中的相关功能作一综述。     

染色质乙酰化相关BRD蛋白家族

Bromodomain结构域首先在果蝇蛋白质Brahma中发现,折叠模式独特且高度保守,是最早也是截至目前公认唯一可与乙酰化赖氨酸结合的结构域。BRD蛋白通过结合不同的蛋白质或者定位蛋白质到细胞核发挥精细调节作用。BRD蛋白复合物常特异性识别并结合到染色质组蛋白H3/H4特定的乙酰化赖氨酸残基,从而影响靶基因的转录翻译;该蛋白复合物功能异常通常与多种疾病的发生相关联,表明对转录翻译调节有重要意义。但迄今为止,BRD蛋白复合物修饰染色质机理不明,现有研究提示BRD蛋白复合物维持染色质乙酰化状态,也可以与染色质组蛋白其它位点结合,从整体水平增强组蛋白乙酰化精度和效率。     

生肌素引起组蛋白H3和H4高度乙酰化及染色质溶解性增加

为研究生肌素在染色质重建过程中的作用 ,采用生肌素真核表达载体转染C2C12肌细胞 ,细胞核经微球菌核酸酶消化后 ,提取DNA进行SDS PAGE分析 .生肌素转染的细胞 ,其核小体 (染色质 )在Mg2 + 溶液中的溶解性明显增加 ,提示染色质组蛋白乙酰化程度提高 .组蛋白经TAU SDS(2 D)双相凝胶电泳分析 ,发现在转染生肌素真核表达载体 2 4h后 ,组蛋白H4的乙酰化修饰程度最高 .采用抗乙酰化组蛋白H3和H4抗体进行的Western印迹分析进一步证明了乙酰化的发生 .上述变化与染色质的活跃程度相关 .RT PCR结果显示 ,生肌素的靶基因烟碱样乙酰胆碱受体 (nAChR)α亚基和肌酸激酶 (MCK)基因在转染后表达水平提高 .结果提示 ,强制性表达外源性生肌素可引起肌细胞核染色质重建 ,进而激活靶基因     

利用果蝇心力衰竭模型筛选果蝇第2号染色体缺失系

果蝇心脏一直以来都是研究心血管发育的极好模型,许多控制心脏分化和特化的调控基因和信号途径从果蝇到哺乳动物都是保守的.由于近年心力衰竭的发病率不断升高,我们最近又建立了果蝇心力衰竭模型用于大规模筛选和鉴定心力衰竭的相关基因.在这个模型中,适龄的成体果蝇被整齐排列在导电的载玻片上,通过电极短暂刺激30s,使果蝇的心跳频率由正常的3Hz增加到6Hz,停止后检测果蝇心率恢复情况,不能恢复正常心跳频率或出现纤维性震颤的果蝇视为心力衰竭.该模型可以在短期内大规模筛选到与心力衰竭相关的基因.利用此心力衰竭模型,我们筛选了164个果蝇2号染色体缺失系,获得33个候选缺失系.这些候选缺失系的心衰率要么与野生型品系相比差异显著,要么与tinman或panier突变系相比差异显著,提示这些缺失系中可能含有与心力衰竭相关的调控基因.     

果蝇基因剂量补偿的实现机制

雌、雄果蝇间的基因剂量补偿是通过雄性果蝇中X染色体相关基因的表达水平上调至雌性果蝇的2倍实现的,基因剂量补偿的实现机制存在2种模型.平衡模型认为是基因组的不一致导致了全基因的反式剂量效应,而MSL复合体将组蛋白修饰酶从常染色体隔离,阻止了常染色体上的基因上调,同时,通过抑制高水平的组蛋白修饰以防止X染色体上的过度补偿....     

影响果蝇心脏发育的基因突变

最近的研究表明,果蝇与脊椎动物及人的心脏早期发育具有极为相似的基因控制机理,果蝇已成为研究人体心脏早期发育基因控制的理想模式动物。利用化学诱变剂甲磺酸乙酯大规模地诱变影响果蝇心脏发育的基因,利用心脏特异性抗体染色进行筛选,获得了112个有心脏突变表型的致死系,其中32个致死系的心脏畸变表型有别于目前已知心脏发育基因的突变表型。细胞遗传学定位研究表明在多线染色体的13个带纹区的某些隐性致死突变基因是目前未知的,其功能可能与发育有关的基因。     

染色质重塑复合物SAGA及其同源物的功能

基因表达调控是生物体生长发育的一个重要环节.在这一过程中,染色质重塑复合物扮演了非常重要的作用.SAGA是一个至少由20个蛋白组成的不依赖ATP的多功能染色质重塑复合物,它通过对组蛋白H3和H2B氨基末端赖氨酸乙酰化修饰来松动染色质结构,从而促进基因转录的起始.目前,对SAGA及其同源物的研究表明,SAGA及其同源物参与了许多重要的生物学功能,如mRNA输出、DNA损伤修复、胚胎发育、细胞癌变等.     

果蝇基因组内的基因剂量补偿效应

基因的剂量补偿最初是指在性染色体性别决定生物中,两性间X染色体数量不同(因而X染色体连锁基因的剂量不同),但基因表达水平基本相同的现象.在非整倍体果蝇中的剂量补偿现象表明,该效应并非仅发生于性染色体上的基因中,在常染色体基因中也普遍存在.综述了X染色体三体及2L染色体三体果蝇在X染色体和常染色体剂量补偿中的最新研究进展...     

组蛋白变体及组蛋白替换

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。对于不同状态的染色质,核小体中会组装入相应的组蛋白变体,并且各种组蛋白变体的尾部也能发生多种修饰。这些变体通过改变核小体的空间构象和稳定性,决定基因转录的激活或沉默,DNA的修复,染色体的异染色化等。在组蛋白替换过程中,组蛋白变体是通过相应的染色质重构复合物组装入核小体,不同的变体有着不同的组装途径。对组蛋白变体的研究是近年来表观遗传学新的研究热点,也是对“组蛋白密码”的新的诠释。并且,组蛋白替换揭示了DNA-组蛋白相互作用变化的一种新的机制。

     

果蝇唾液腺染色体观察实验的改进

《全日制中学生物学教学大纲》明确规定 :使学生掌握关于染色体、DNA和基因三者之间的相互关系的知识以及基因的概念。笔者在高中生物学教学中利用果蝇三龄幼虫作实验材料 ,并对该实验步骤进行了一些探索和改进 ,在指导学生理解和掌握染色体、DNA与基因的相互关系的知识方面 ,取得了较好的教学效果 ,具体作法如下 :1　实验原理染色体的数量和形态特征跟生物体的性状遗传有着密切的关系 ,而染色体的形态特征又不易观察 ,科学家们发现果蝇三龄虫的唾液腺细胞处于永久早期 ,染色体解旋呈伸展状态。在幼虫发育过程中细胞核中的DNA多次复制 ,…     

CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory

DNA synthesis during S-phase and upon DNA repair is accompanied by chromatin assembly. The chromatin assembly factor CAF-1 has been biochemically well-characterized to deposit histones onto newly synthesized DNA. To gain insights into the in vivo functions of CAF-1 in Drosophila, we generated null mutants of the largest subunit of dCAF-1, dCAF-1-p180. We show that, unlike CAF-1 mutant yeast, dCAF-1-p180 mutant flies are hemizygous lethal. Removal of maternal dCAF-1-p180 activity by germline clones blocks oogenesis. Tissue-specific deletion of dCAF-1-p180 in the eye primordia disrupts eye development. In addition, reduction of dCAF-1-p180 activity suppresses gene silencing at heterochromatin and antagonizes Polycomb-mediated cell fate determination. Furthermore, heterozygous dCAF-1-p180 mutant flies display an increased sensitivity to γ-irradiation and a reduced efficiency in recombinational double strand break (DSB) repair. Our experiments also show that human hCAF-1-p150 can rescue the dCAF-1-p180 mutant flies, demonstrating a functional conservation of eukaryotic CAF-1 activities in vivo. Together, our results establish that dCAF-1-p180 is an essential gene for Drosophila development and further underscore the importance of dCAF-1 in regulating gene expression and DNA repair in vivo.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

我国原生植物假泽兰(菊科)的细胞学研究

该文研究了我国原生植物假泽兰(Mikania cordata)台湾花莲居群、苗栗居群、宜兰居群以及台北居群的染色体数目和染色体形态。结果表明:所有居群的染色体数目为2n=36,第一对染色体为近中部着丝粒染色体,其长臂中部具有次缢痕,显著大于其余染色体。各居群的核型公式皆为2n=18m+18sm,核型均为2B型,染色体内不对称性指数(A_1)的变化范围为0.38~0.39,染色体之间不对称性指数(A_2)的变化范围为0.30~0.32。此为我国假泽兰居群染色体数目的唯一报道,也是对该种核型的首次报道。结合前人对假泽兰染色体数目的研究结果,认为假泽兰存在种内非整倍性现象,但在中国台湾的居群中目前仅发现基于x=18的二倍体(2n=36)。假泽兰的第一对染色体的长臂中部具次缢痕,与假泽兰属已报道的核型相似,这一次缢痕可作为假泽兰属的细胞学标记。核型资料、野外观察以及ISSR数据显示薇甘菊(M.micrantha)在我国的成功入侵与入侵种和本土种之间的杂交渐渗无关。根据标本记录和野外考察结果,我国假泽兰现在的分布区与过去相比有了很大的缩减,推测生境的破坏和薇甘菊的侵入可能是导致假泽兰在中国台湾地区逐渐消失的主要原因。     

Evidence for recombination and segregation of virulence to pine in a hybrid cross between Gibberella circinata and G. subglutinans

Two species associated with the Gibberella fujikuroi species complex, G. circinata (the cause of pitch canker in pines) and G. subglutinans (avirulent on pine), were found to have limited interfertility in hybrid crosses. MAT idiomorphs, polymorphisms in the histone H3 gene, vegetative compatibility, and virulence phenotypes were used to verify recombination. The MAT idiomorphs appeared to be assorting independently, but the histone H3 haplotype was disproportionately represented by that of the G. subglutinans parent. Ninety-eight percent (45/46) of the progeny tested were vegetatively incompatible with both parents. All F₁ progeny were avirulent to pine, but a wide range of virulence was restored through a backcross to the virulent parent (G. circinata). Attempts at hybrid crosses using other isolate combinations were rarely successful (1/26). This limited interfertility supports retention of G. circinata and G. subglutinans as separate species, but offers opportunities to characterize the inheritance of virulence to pine.     

半蒴苣苔属植物染色体制片优化及染色体数目和倍性研究

染色体数目和倍性是系统与进化生物学和遗传学研究中十分重要的基础信息。为探索半蒴苣苔属染色体制片的适宜条件以及染色体数目的进化模式及其与物种的进化关系，该研究基于半蒴苣苔属染色体数目的进化历史，并根据该属植物具有叶片扦插繁殖的特性，采用叶片水培生根法获取半蒴苣苔(Hemiboea subcapitata)、弄岗半蒴苣苔(H.longgangensis)、龙州半蒴苣苔(H.longzhouensis)、江西半蒴苣苔(H.subacaulis var.jiangxiensis)、华南半蒴苣苔(H.follicularis)和永福半蒴苣苔(H.yongfuensis)6种植物的根尖材料，分析不同实验条件对染色体制片效果的影响，对染色体制片实验的条件进行优化及染色体计数，结果表明：(1)9:30—10:00取材，解离10 min以及染色15 min为半蒴苣苔属染色体制片的适宜条件。(2)上述6种半蒴苣苔属植物均为二倍体，染色体数目均为32(2n=2x=32)。(3)除个别物种染色体数目有变化以外，该属大部分物种染色体数目可能为2n=2x=32且染色体数目变化可能是非整倍化的作用，与物种进化没有明...     

In vivo analysis of <Emphasis Type="Italic">Drosophila</Emphasis> SU(<Emphasis Type="Italic">Z</Emphasis>)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.     

Replication-independent histone deposition by the HIR complex and Asf1

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.     

横断山区六种八居群鼠尾草属植物的核型分析

鼠尾草属(Salvia)是唇形科(Lamiaceae)最大的属,属下多种为民间常用草药,亦有供观赏的种类。为探究横断山区物种在细胞学水平的进化方式,讨论形态分类学与分子系统学之间的分类关系,该研究通过广泛收集染色体文献资料,采用植物常规压片法对采集自横断山地区6种8居群鼠尾草属植物进行核型分析,并构建了中国地区分布的鼠尾草属植物叶绿体系统发育树。统计结果表明:(1)全世界范围内报道了约23%的鼠尾草属植物染色体数据,其中分布在中国地区的鼠尾草属植物染色体报道率为32.10%,分布在横断山地区的鼠尾草属植物报道率为40.54%,(2)鼠尾草属植物染色体基数以x=8和x=11为主,分布在中国地区的鼠尾草属植物染色体基数均为x=8。实验结果表明:(1)西藏鼠尾草(S. wardii)核型数据为首次报道。(2)雪山鼠尾草(S. evansiana)首次在云南德钦地区发现二倍体居群。将细胞学数据结合叶绿体进化树开展染色体进化关联分析,论证多倍化可能不是鼠尾草属物种适应高海拔环境的主要机制,表明多倍体不是该属物种形成的主要进化途径而是以二倍体水平为主,推测染色体组的加倍可能是物种在形态学与分子系统学上分类关系不一致的原因之一。该研究丰富了横断山区鼠尾草属植物的染色体核型数据,结合区域分子系统树探讨染色体特征的进化关系,为今后深入研究该属物种的核型进化做出了探索,为开展祖先物种染色体基数推演分析补充了基础数据。     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.