用计算机对人类TSPY1基因P53结合位点的鉴定

根据p53下游基因在其调节区域（启动子或内含子）含有与P53蛋白特异性结合的一致性序列5’ –RRRCWWGYYYN（013) RRRCWWGYYY3’,R=G或A,W=T或A,Y=C或T,N=A,C,T,G。用计算机对人类基因组中P53结合位点进行了研究,发现Y染色体上的TSPY1基因内含子中含有这样的一致性序列5’GGGCTAGTTTtgGAGCTAGCCT3’,意味着TSPY1基因有可能是一个p53下游基因。     

人类P53下游基因一致性序列研究

人类P53蛋白是一种通用转录因子，通过调控一系列下游基因的转录来影响许多细胞功能。p53下游基因含有P53蛋白结合序列，收集已报道的63条人类P53蛋白结合序列并与E1-Deiry等定义的一致性序列进行比较，发现这些P53蛋白结合序列与一致性序列特征并不严格一致，对这些偏差规律的分析有利于建立p53下游基因预测模型，进而利用计算机方法预测p53下游基因，研究其基因互作网络。     

人类P53下游基因一致性序列研究

人类P53蛋白是一种通用转录因子,通过调控一系列下游基因的转录来影响许多细胞功能。p53下游基因含有P53蛋白结合序列,收集已报道的63条人类P53蛋白结合序列并与ElDeiry等定义的一致性序列进行比较,发现这些P53蛋白结合序列与一致性序列特征并不严格一致,对这些偏差规律的分析有利于建立p53下游基因预测模型,进而利用计算机方法预测p53下游基因,研究其基因互作网络。     

乐至黑山羊GH和IGF-I基因多态性及其与产羔性状的关联性分析(英文)

生长激素(GH)胰岛素样生长因子I(IGF-I)在卵泡的生长和闭锁中发挥关键作用。研究采用PCR-SSCP技术对157只乐至黑山羊GH和IGF-I进行多态位点的检测。结果发现,GH基因5’侧翼区和第二外显子分别检测到2种基因型,第三外显子检测到4种基因型,第四和第五外显子分别检测到3种基因型;IGF-I基因的第二外显子检测到2种基因型。通过基因测序发现GH基因有14处单核苷酸突变位点(SNPs)—T48C,A55G)(P1引物),T781C(P2引物),G1122A,A1161G,A1171G,C1179T和C1180G(P3引物),T1481C,A1494G,G1549T和C1590T(P4引物),G1942A和G1957A(P5引物);IGF-I基因有1个SNP(G3270C)(P6引物)。关联性分析结果表明,只有GH基因第三外显子BB型乐至黑山羊产羔数比AA型多0.32(p0.05),其余基因型间产羔数差异不显著。研究初步表明GH基因可能与乐至黑山羊产羔数之间存在一定关联性。     

基于植物p53保守序列构建RNAi的DNA片段方法

构建p53基因RNA干涉DNA片段的目的,是将其应用于植物悬浮培养中的细胞周期调节.依据拟南芥p53基因与其他高等植物具有高度保守区域的特点,合成其两个区域的核苷酸片段(Ⅰ和Ⅱ)及其相应的反向片段(Ⅰ′和Ⅱ′),并在Ⅱ和Ⅱ′端部分别引入内含子两个端部核苷酸序列.在相互连接后进行PCR选择扩增,其产物再与克隆载体连接并经过蓝白斑筛选获得重组DNA;在电泳和核苷酸测序鉴定后表明,最终得到了Ⅰ–5′–内含子–Ⅱ′–Ⅱ–内含子–3′–Ⅰ′序列的重组DNA片段.该片段两端含多克隆位点,通过插入植物的表达载体进入细胞基因组,在细胞中其转录产物将形成发夹结构,经胞内酶切后可以形成短的双链RNA片段,将具有干涉p53基因表达的功能.     

GCK2软件在p53基因克隆过程中的应用

p53基因是迄今发现与人类肿瘤相关性最高的押癌基因，通过克隆p53基因对其表达调控进行研究。利用分子生物学软件GCK2．0对整个克隆过程进行分析，制定克隆策略，通过测序鉴定结果，得到了舍目的基因p53cDNA全序列的pTRE—p53重组质粒。通过GCK2．0分析，减少了基因克隆的盲目性，提高了工作效率。     

慢性粒细胞白血病急变与bcr／abl，p53,p16基因相关性研究

应用RT-PCR、PCR-SSCP方法对33例慢性粒细胞白血病（Chronic myeloid leukemia,CML）进行了bcr/abl,p53,p16基因检测，发现33例CML中32例bcr/abl基因阳性（97.0%）。22例慢性期无p53基因突变，而11例急变组中一例外显子5和一例外显子7发生p53基因突变，且均发生于急粒变组，2例出现p16基因纯合缺失，且均发生在急淋变组，33例CML均未见p16基因突变，结果提示：bcr/abl基因与CML发生有关，而p53基因突变可能与CML急粒变有关，p16纯合缺失可能与CML急淋变有关。     

淫羊藿素对不同p53分型顺铂耐药卵巢癌细胞株周期阻滞作用的研究

目的:研究淫羊藿素(ICT)对不同p53分型顺铂耐药卵巢癌细胞株的增殖抑制作用及机制,为复发耐药性卵巢癌治疗药物的研发提供新思路.方法:利用胸腺嘧啶核苷酸(TdR)双阻滞法建立周期同步化模型,采用流式细胞技术检测淫羊藿素对同步化后的不同p53分型顺铂耐药卵巢癌细胞株的细胞周期阻滞作用,Western blot检测浓度为20μmol/L淫羊藿素作用下细胞株周期蛋白CyclinE、CyclinB1、p53及CHK1蛋白表达情况.结果:淫羊藿素浓度为20μmol/L作用4 h时即产生周期阻滞作用,阻滞效果随作用时间延长而增强;其中,p53野生型C13~*细胞株G1期百分比升高(P0.05);p53变异型A2780cp细胞株G2期百分比升高(P0.05);p53缺失型的SKOV3细胞株G2期百分比升高(P0.05).淫羊藿素20μmol/L作用下,p53野生型C13~*细胞株CyclinE表达下调,而CyclinB1蛋白无明显变化;p53变异型A2780cp及p53缺失型SKOV3细胞株CyclinB1蛋白表达下调,而CyclinE无明显变化.p53野生型C13~*细胞株p53蛋白表达明显上调,p53变异型A2780cp细胞株p53蛋白无明显变化,p53缺失型SKOV3细胞株p53蛋白表达缺失;三株细胞CHK1蛋白表达均明显升高.结论:淫羊藿素对不同p53分型顺铂耐药卵巢癌细胞均具有时间依赖性的周期阻滞作用;其对不同p53分型顺铂耐药卵巢癌细胞的阻滞作用途径不同;其中对p53野生型C13~*细胞株通过p53途径下调CyclinE蛋白诱发细胞G1期阻滞;而对p53突变型A2780cp及p53缺失型SKOV3细胞株则通过CHK1途径下调CyclinB1诱发G2期阻滞.     

猪生长激素基因启动子区的序列变异研究

采用聚合酶链式反应(PCR)及PCR产物直接序列分析方法，对猪生长激素基因(pGH)5’-侧翼区(启动子区)序列的遗传变异进行研究，共分析了6头临高猪、3头玉山黑猪和5头蓝塘猪．结果表明：在猪生长激素基因启动区，共有10处碱基替换，它们分别位于：72位C→T，238位G→T，343位A→G，274位A→G，721位G→C。722位T→C，723位G→C，728位C→T，784位G→C，787位G→T．2处碱基插入，依次是180位插入碱基G。416位插入碱基C．新发现了3处碱基置换，分别是721位G→C，722位T→C，723位G→C和1处碱基插入，416位插入碱基C．     

一类核糖体蛋白基因碱基出现频率分析

选取69个含内含子的核糖体蛋白基因,抽取其中每个基因转录起始位点附近长度为100个碱基的序列,分析69个基因序列样本碱基A,T,G,C出现的频率,发现碱基出现频率最大的为A,其次是T。含内含子的核糖体蛋白基因中富含碱基A,T的序列可能有利于基因的转录。     

A role for branchpoints in splicing in vivo

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

High frequency of p53 intronic point mutations in laryngeal squamous cell carcinoma

Intronic point mutations are rare and totally unknown for human laryngeal squamous cell carcinoma (LSCC). To explore the relationship of p53 gene intronic mutation to the development of human LSCC, DNA was extracted from both tumor tissues and matched normal tissues of 55 patients with LSCC in northeast of China. Polymerase chain reaction amplification-single strand conformational polymorphism (PCR-SSCP) combined with silver staining and DNA direct sequencing were used to detect mutations in exons 7～8 (p53E7 and p53E8) and introns 7～8 (p53I7 and p53I8) of p53 gene. The p53E7 mutation was detected in 17 out of 55 patients, and the p53I7 mutation in 21 patients. No mutation was found at p53E8 or p53I8 site. The difference between tumor group and paired normal group on the rates of both p53E7 and p53I7 mutations was statistically significant. The rate of p53I7 mutations in tumor tissue was higher than that of normal tissue, and so was that of p53E7. Sequence analysis revealed that most p53I7 mutations were at the nucleotides in the branch point sequence or the polypyrimidine tract in the 3′-splice acceptor site of the intron 7. The high incidence of p53 gene intronic mutation in LSCC indicates that genetic changes within the noncoding region of the p53 gene may serve as an alternative mechanism of activating the pathogenesis of human laryngeal squamous cell carcinoma. Mutations in the noncoding region of this gene should be further studied.     

GCK2软件在p53基因克隆过程中的应用

p53基因是迄今发现与人类肿瘤相关性最高的抑癌基因,通过克隆p53基因对其表达调控进行研究。利用 分子生物学软件GCK2.0对整个克隆过程进行分析,制定克隆策略,通过测序鉴定结果,得到了含目的基因 p53cDNA全序列的pTRE-p53重组质粒,通过GCK2.0分析,减少了基因克隆的盲目性,提高了工作效率。     

利用文本数据挖掘研究p53基因表达调控网络

为了研究p53基因与其下游/靶基因的关联性,以了解p53基因表达调控网络,采用文本数据挖取方法,利用自编的Perl 5.10程序,对PubMed文献数据库中p53基因相关文献及人类基因本体数据库进行数据挖掘,并利用连锁聚类法构建p53基因表达调控网络图.结果发现,目标基因的频率分布同文本中所有基因本体的频率分布存在一定的关联性,低频基因的文本挖掘比例明显低于高频基因的文本挖掘比例.从而说明,p53基因表达调控网络中各基因的分布情况与基因频率有较大关系,而文本数据量对文本数据挖掘的准确率也有重要影响.