The hepatitis C virus 5’ untranslated region gene amplified by rapid amplification of cDNA ends and its secondary structure

Objectives: To obtain very end full-length cDNA ofhepatitis C virus (HCV) 5’ untranslated region(5’UTR) and analyze its primary and secondarystructure.Methods: A patient infected genotype 2a HCV wasidentified by reverse transcription-nested polymerasechain reaction (RT-PCR) and restriction fragmentlength polymorphism (RFLP). Total RNA isolatedfrom the serum was used as template, and the cDNAof the 5’ untranslated region was amplified using rap-id amplification of cDNA ends (RACE). The frag-ments were recombinated by A-T clone strategy, andthe recombinants were confirmed by RFLP andPCR, and sequenced subsequently. Secondary struc-tures were analysed by RNAdraw.Results: Very end full-length cDNA of genotype 2aHCV 5’ UTR was obtained by RACE. In five clonesobtained, three contained full-length 5’UTR cDNA;A21G, G170A, T222C, T247C, C339T substitutionswere found as compared to HC-J6. Homological re-sults of HCV-1, HC-J6, HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions didnot alter secondary structure. Two of 5 clones weredeletions of 53bp and 135bp at the 5’terminal ofHCV 5’UTR, respectively.Conclusions: RACE can be used to obtain the full-length cDNA of 2a genotype HCV 5’UTR. Genes de-leted at the 5’ terminal of HCV circulate in hepatitisC patients.

丙型肝炎5'非编码区末端快速基因扩增方法的建立及应用

目的建立快速获得丙型肝炎(HCV)基因组5非编码区(5 UTR)真末端序列的分子生物学方法。方法逆转录后利用末端聚合酶(TDT)进行加尾反应,再利用套式聚合酶链反应(PCR)扩增出目的末端基因的cDNA片段,A-T克隆,用限制性内切酶片段长度多态性分析(RFLP)与PCR鉴定重组子,全自动序列分析仪测定插入子序列。结果cD-NA末端快速扩增技术(RACE)获得5株HCV5 UTR克隆,包括3株全长克隆和2株缺失克隆。2株缺失克隆,一条在5末端缺失53个碱基,另一条缺失144个碱基。结论RACE技术快速、有效、实用,可有效获得丙型肝炎病毒基因组的5非编码区末端序列。

四种实验用小型猪mtDNA控制区5＇端序列的比较研究

目的测定西藏小型猪以及巴马小型猪、贵州香猪和五指山猪的mtDNA控制区5’端序列，比较研究其遗传变异和与国内家猪的亲缘关系。方法扩增四种小型猪的mtDNA控制区5’端，测序并进行所测序列多重比对，确定变异位点、单倍型，并建立亲缘关系树。结果与荣昌猪相比，西藏小型猪mtDNA控制区5’端侧翼区704bp，有20个变异位点，由此归纳出26个单倍型；其中有两种主体单倍型，三个转换位点（305，500，691）的碱基分别为t，a，a和c，g，g。巴马小型猪、贵州香猪和五指山猪mtDNA控制区5’端变异位点较少，分别只有4、4、3种单倍型。亲缘关系树的分析表明，西藏小型猪与其它三种小型猪有较近的亲缘关系。结论西藏小型猪群体存在遗传分化，与巴马小型猪、贵州香猪和五指山猪亲缘关系较近；巴马小型猪、贵州香猪和五指山猪遗传均一性较高，可能与长期的人工选择有关。

植物花器官的基因表达

本文从基因水平上概述了植物花的分化、发育研究的现状和进展,从花器官早期发育基因、花瓣特异性基因和雌雄蕊特异性基因的表达等的研究现状可知,调控花器官特异性基因表达的元件主要分布于基因的5′端启动子区域,该区域可使嵌合基因在异源植物中正确表达。

Higher Concentrations of Glucose or Insulin Increase the Risk of Various Types of Cancer in Obesity or Type 2 Diabetes by Decreasing the Expression of p27Kip1, a Cell Cycle Repressor Protein

Research Aims: Obesity and type 2 diabetes are known to be associated with increased risk of various types of cancer. However, the molecular biological mechanism of how the risk of cancer is increased in obesity or type 2 diabetes is not known. The aim this research is to investigate if the decreased expression of p27Kip1, a cell cycle repressor protein, plays a central role in this mechanism. Research Methods, Previous Studies and Theoretical Backgrounds: It is well known that the expression of p27Kip1 is increased by numerous nutritional or chemopreventive anti-cancer agents. But it has never been known that the expression of p27Kip1 could be decreased, rather than increased, and the risk of cancer could be increased, rather than decreased. This problem was solved recently and this new analytical method was used in this study. Results: 1) The expression of p27Kip1 was indeed significantly decreased in human obese type 2 diabetic individuals relative to the lean normal controls. 2) The expression of p27Kip1 was also significantly decreased in genetically obese rodents relative to the lean normal controls. Additionally, in obese rodents, the concentrations of glucose or insulin were significantly increased relative to the lean normal controls. 3) Using human cells cultured in vitro it was found that the increased concentrations of glucose or insulin decrease the expression of p27Kip1. Conclusions: These results suggest that higher concentrations of glucose or insulin increase the risk of various types of cancer in obesity or type 2 diabetes by decreasing the expression of p27Kip1.

2a型丙型肝炎病毒5′非编码区的RACE扩增及二级结构的分析

目的 获得真全长中国大陆 2a型丙型肝炎病毒 (HCV) 5′非编码区 (5′UTR)cDNA ,并分析其一级结构和二级结构 ,为进一步研究其在HCV复制、翻译中的调控机制、开发新的抗HCV药物奠定基础。方法 利用逆转录套式聚合酶链反应 (RT PCR)联合限制性内切酶长度多态性分析(RFLP)初步筛选出 1例 2a型HCV感染者 ,采用cDNA末端快速扩增技术 (RACE)扩增出一条约 80 0bp的cDNA片段 ,A T克隆 ,用RFLP与PCR鉴定重组子 ,全自动序列分析仪测定插入子序列 ,RNAdraw预测二级结构。结果 RACE法获得真全长 2a型HCV 5′端序列。 5个克隆中 ,3个克隆含全长 5′UTR序列 ,与HCV 1,HC C2 ,HC J6 ,HC J8相比 ,同源性分别为 93.6 %～ 94 .4 % ,92 .1%～ 93.0 % ,98.8%～99 .7% ,96 .2 %～ 96 .5 % ,与 2a型标准株HC J6相比 ,2 1,170 ,2 2 2 ,2 4 7,339位不同 ,分别为G ,A ,C ,C ,T ,但这些突变不影响其二级结构。另外 2个克隆为 5′端缺失突变株 ,分别缺失 5 4bp和 14 4bp。结论 RACE技术快速、有效、实用 ,可有效获得病毒基因组的末端序列 ;依此获得中国大陆 2a型HCV的 5′UTRcDNA ;在感染者血液中存在 5′端部分缺失的HCV基因片段。