一种新型锌指蛋白基因——人ZNF313的克隆、定位及表达(英文 )

运用正常可育男性和无精症病人睾丸组织的mRNA差异显示和cDNA末端快速扩增 (RACE)等方法 ,从人睾丸组织中分离了一个同时含有指环结构和C2 H2 结构域的新型锌指蛋白基因———人ZNF3 13。运用荧光原位杂交 (FISH)方法 ,将该基因定位到人染色体 2 0q13。该基因含 6个外显子 ,编码 2 2 8个氨基酸。基因组结构分析显示 ,外显子 6含有的 2个加尾信号 ,产生 2种不同的 3′端非翻译区。Northern杂交及多组织RT PCR的结果显示该基因含有 0 .75kb和 2 .4kb两种转录本 ,其中0 .75kb转录本在正常睾丸中高表达 ,而其他组织、无精症患者及胎儿睾丸组织中该基因低表达。结果提示 :人ZNF3 13基因对精子发生和男性可育性可能起重要作用     

RING Finger Protein RNF207, a Novel Regulator of Cardiac Excitation

Two recent studies (Newton-Cheh, C. et al. (2009) Common variants at ten loci influence QT interval duration in the QTGEN Study. Nat. Genet. 41, 399–406 and Pfeufer, A. et al. (2009) Common variants at ten loci modulate the QT interval duration in the QTSCD Study. Nat. Genet. 41, 407–414) identified an association, with genome-wide significance, between a single nucleotide polymorphism within the gene encoding RING finger protein 207 (RNF207) and the QT interval. We sought to determine the role of RNF207 in cardiac electrophysiology. Morpholino knockdown of RNF207 in zebrafish embryos resulted in action potential duration prolongation, occasionally a 2:1 atrioventricular block, and slowing of conduction velocity. Conversely, neonatal rabbit cardiomyocytes infected with RNF207-expressing adenovirus exhibited shortened action potential duration. Using transfections of U-2 OS and HEK293 cells, Western blot analysis and immunocytochemistry data demonstrate that RNF207 and the human ether-a-go-go-related gene (HERG) potassium channel interact and colocalize. Furthermore, RNF207 overexpression significantly elevated total and membrane HERG protein and HERG-encoded current density by ∼30–50%, which was dependent on the intact N-terminal RING domain of RNF207. Finally, coexpression of RNF207 and HSP70 increased HERG expression compared with HSP70 alone. This effect was dependent on the C terminus of RNF207. Taken together, the evidence is strong that RNF207 is an important regulator of action potential duration, likely via effects on HERG trafficking and localization in a heat shock protein-dependent manner.     

运用数字差异展示方法克隆一个人类新的锌指蛋白基因ZNF474

运用数字差异展示方法,克隆一个与生精相关的睾丸高表达基因。借助公共ESTs数据库,利用DDD软件比较分析各种睾丸文库与其他组织或细胞系文库有差异表达的ESTs,成功克隆到一个在人类睾丸中高表达的新基因。结合实验获得新基因cDNA全长,该基因被国际人类基因命名委员会命名为ZNF474(GeneBank登陆号AY461732)。ZNF474的cDNA全长为1 972 bp,定位在5 q23.2。通过RT-PCR及测序验证,其开放阅读框的位置在377 bp~1 471 bp处,编码364个氨基酸,在氨基酸水平与小鼠同源基因有66%的一致性,而与其他已知蛋白质无明显同源性。Northern杂交分析显示ZNF474在成体睾丸组织特异高表达,卵巢组织弱表达,在多种其他组织中不表达,为单一转录本。原位杂交显示ZNF474基因在正常成人睾丸组织各级生精细胞、隐睾组织以及精原细胞癌组织中均有较高表达。综上考虑,推测ZNF474作为生殖细胞中特异的转录因子,对人类的精子发生和卵母细胞的发育可能起重要作用。     

一个麻疯树RING型锌指蛋白基因JcRFP1的克隆及表达

首次从麻疯树胚乳cDNA丈库中克隆得到一个RJNG型锌指蛋白基（GenBank登录号为JF920726）,命名为JcRFP1。该cDNA长度为728bp,包含编码JcRFP1蛋白的完整开放阅读框（516bp）。脚,基因在麻疯树各器官中均检测到表达且表达量依次为：叶〉茎〉花〉果实〉种子〉根。将克隆到的JcRFP1基因的cDNA序列连接到表达载体pET32a（＋）上,导入BL21（DE3）pLysS菌株,成功诱导表达相对分子质量为33．2kDa的可溶性融合蛋白。该融合蛋白免疫新西兰大白兔,得到效价为1：6500的抗血清。研究表明,JcRFP1蛋白具有体外泛素连接酶E3活性,在麻疯树体内可能参与油菜素甾醇信号转导途径。     

一个新的人类锌指蛋白基因ZNF474的cDNA克隆和表达分析

数据库消减杂交就是利用NCBI中的Unigene数据库中大量的数据资源,收集各种细胞或组织的基因表达谱进行两两比较或多重比较,获得两组文库间有统计学意义的差异表达基因。运用NCBI中的数据库消减杂交分析方法,从人睾丸组织中分离了一个含有C2H2结构的新型锌指蛋白基因-ZNF474(GenBank登录号：AY461732)．通过推导和进一步的RT—PCR实验证实：该基因含2个外显子,gDNA在染色体上跨度30065bp,定位于人染色体5q23．1-q23。2．cDNA编码一个含364个氨基酸的新蛋白,分子质量是40．3kDa,等电点为9．59。Northem杂交结果显示：该基因含有2．37kb大小的唯一转录本,主要在睾丸中很强表达,卵巢中有弱表达,而其他组织中该基因无表达．结果提示：ZNF474基因对精子发生和卵母细胞的发育可能起重要作用、     

RMA1 an Arabidopsis thaliana Gene Whose cDNA Suppresses the Yeast secl5 Mutation, Encodes a Novel Protein with a RING Finger Motif and a Membrane Anchor

To identify molecules that function in the plant secretory pathway,we screened for Arabidopsis thaliana cDNA clones that complementthe temperature-sensitive (ts), secretion-deficient sec15 mutationof yeast Saccha-romyces cerevisiae. RMA1, one of the genes obtainedin this screening, suppressed not only the ts growth of sec15but also its secretory defect. RMA1 is not a structural homologueof SEC15 but encodes a novel 28 kDa protein with a RING fingermotif and a C-terminal membrane-anchoring domain. Mutationalanalysis indicates that the RING finger motif of RMA1 is importantfor its suppression activity. In Arabidopsis plant, RMA1 isubiquitously expressed. A search for homologous proteins inthe database revealed that Arabidopsis, nematode, mouse andhuman possess close homologues of RMA1. (Received January 5, 1998; Accepted March 9, 1998)     

编码锌指蛋白的人类新基因TFL76的电子克隆

目的：根据基因同源同功原理电子克隆人类新基因。方法：利用基于基因识别软件Genescan和EST拼接的同源基因克隆法得到人类新基因序列TFL76,再利用生物信息学数据库和软件对其进行功能的预测和分析。结果：TFL76的cDNA序列长2268bp,开放阅读框编码677个氨基酸残基,含12个连续的C2H2型锌指基序,其分子量为76kDa。编码区序列被4个内含子分割。染色体定位于19q13．4。此位点存在很多与胃癌、膀胱癌、乳腺癌等癌症相关的基因。TFL76的N末端含有多种蛋白激酶的磷酸化位点和核定位信号。结论：TFL76可能是一个和癌症相关的核转录因子。     

Identification of a Deubiquitinating Enzyme as a Novel AGS3-Interacting Protein

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.     

沙冬青RING型锌指蛋白基因AmRFP1的克隆与功能初步分析

利用RT-PCR方法,从强抗逆植物蒙古沙冬青克隆到1个受寒旱诱导的锌指蛋白基因AmRFP1。预测其编码蛋白由366个氨基酸残基组成,其中含有1个C3H2C3型锌指结构域,故属于RING-H2(C3H2C3)型锌指蛋白。该蛋白还含有2个跨膜区,很可能定位于细胞质膜。半定量RT-PCR分析表明,在不同季节野外生长沙冬青植株嫩叶中,AmRFP1在夏季只有低水平表达,进入秋季后表达量明显增加,尤其在秋末冬初其表达量迅速增加并达到全年最高峰,但在进入最寒冷的隆冬后又回落至秋季水平并维持到翌年春季基本未变。将该基因在拟南芥中超表达,则明显降低了转基因拟南芥的抗冻性。     

苹果一个锌指蛋白基因的cDNA克隆及其表达特性分析(英文)

A cDNA library was created from stem apex tissue from Jonathan apples (Malus domestica Borkh.), harvested in June to August, during which the plant transitions from vegetative growth to reproductive growth. From this library, we isolated an expressed sequence tag (EST) sequence containing a zinc finger motif, using this sequence, a 779 bp cDNA fragment was obtained by using 5‘ RACE, and a final full-length cDNA encoding an apple zinc finger protein (named MdZF1; GenBank accession number AB116545) was obtained by further PCR. This zinc finger motif of MdZF1 has high homology with INOETERMINATE1 (ID1) gene from maize which seemed to be involved in the transition to flowering. Northern blot and RT-PCR analyses showed that the MdZF1 expressed in the root, stem, leaves, shoot apex and floral organs of the apple, with expression levels higher in root, stem, leaves and floral shoot apex than that in floral organs (sepals, petals, stamens and pistils). Genomic Southern analysis showed that there was a single copy gene in apple genome.