丙型肝炎病毒准种在NS2区的变异状况初探

探讨HCV准种在NS2区的基因结构特征及变异状况。利用逆转录－巢式PCR从1份HCV慢性携带者的阳性血清及1份丙肝患者的血清中获得HCV NS2全长cDNA,将其克隆于T载体,各随机挑取5个阳性克隆进行序列测定,结果显示克隆到HCV NS2全长基因,所测克隆在核苷酸水平和氨基酸水平互不相同。该慢性携带者HCV NS2区序列以完整读码框架（ORF）为主,一个于HCV多聚蛋白第835位氨基酸的位置出现终止信号,而该丙型肝炎患者以NS2N端发现终止信号的序列为主,其中三个于第835位氨基酸的位置出现终止信号,一个于第887位氨基酸的位置出现终止信号,仅一个克隆的序列为完整ORF。对ORF完整的序列进行比较,发现丙型肝炎患者氨基酸变异主要集中于N端,蛋白二级结构模拟显示丙肝患者NS2与慢性携带者的优势二级结构类似,研究表明从我们选择的两种感染者的HCV NS2序列看,不同临床类型的HCV病人体内的HCV准种在NS2区存在差异,这种差异可能与病毒存在于机体的状态一定的一致性。     

丙型肝炎病毒北京株NS3基因的克隆及测序

用ＲＴ－ＰＣＲ方法从北京株丙型肝炎病毒中扩增出ＮＳ３区基因片段,该片段经ｐＧＥＸ－Ｔ载体克隆到大肠杆菌ＤＨ５α菌株中．经自动序列分析仪测出ＮＳ３基因的７２８ｂｐ长的核酸序列．发现在该片段中第３１位核苷酸发生Ａ→Ｔ突变,产生一个终止突变．这表明,丙型肝炎病毒北京株存在一定的变异,产生缺陷型病毒,这类缺陷型病毒的出现可能是丙型肝炎病毒持续性感染的原因之一．     

Evolution of Prokaryotic Genes by Shift of Stop Codons

De novo origin of coding sequence remains an obscure issue in molecular evolution. One of the possible paths for addition (subtraction) of DNA segments to (from) a gene is stop codon shift. Single nucleotide substitutions can destroy the existing stop codon, leading to uninterrupted translation up to the next stop codon in the gene’s reading frame, or create a premature stop codon via a nonsense mutation. Furthermore, short indels-caused frameshifts near gene’s end may lead to premature stop codons or to translation past the existing stop codon. Here, we describe the evolution of the length of coding sequence of prokaryotic genes by change of positions of stop codons. We observed cases of addition of regions of 3′UTR to genes due to mutations at the existing stop codon, and cases of subtraction of C-terminal coding segments due to nonsense mutations upstream of the stop codon. Many of the observed stop codon shifts cannot be attributed to sequencing errors or rare deleterious variants segregating within bacterial populations. The additions of regions of 3′UTR tend to occur in those genes in which they are facilitated by nearby downstream in-frame triplets which may serve as new stop codons. Conversely, subtractions of coding sequence often give rise to in-frame stop codons located nearby. The amino acid composition of the added region is significantly biased, compared to the overall amino acid composition of the genes. Our results show that in prokaryotes, shift of stop codon is an underappreciated contributor to functional evolution of gene length.     

一株丙型肝炎病毒缺陷株NS5A基因片段的序列分析

Ｑ丙型肝炎是由丙型肝炎病毒（ＨＣＶ）引起的一种严重的传染病。丙型肝炎病毒主要通过输血和应用不洁的血液制品传播,所以利用敏感、特异的检测方法筛选献血员对丙型肝炎的预防尤为重要。现在第二代ＨＣＶ检测试剂已大批应用,加入ＮＳ５Ａ抗原的第三代试剂也正在研制中...     

Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

The development of transgenic mice for the expression of large amounts of human lysozyme in milk

Human lysozyme (hLYZ) has important potential applications as antimicrobial medicine and food additive. To develop a robust expression vector that ensures expression of large amounts of hLYZ in milk, here a 26,267 bp chimeric mouse whey acidic protein (mWAP)::hLYZ cassette was constructed and used as a mammary gland-specific expression vector, in which a 3,010 bp genomic sequence in the 24,466 bp mWAP gene locus was substituted by a 4,811 bp genomic sequence of hLYZ, exactly from the start codon to the stop codon. Corresponding transgenic mice were generated, and enzymatically-active hLYZ was expressed at 18.4–35 g l^?1 in the milk of most transgenic mouse lines. Our transgenic mice carrying chimeric mWAP::hLYZ represent a model system for cost-effective production of hLYZ.     

丙型肝炎核心蛋白与NS3蛋白在转基因小鼠中的表达

为研究丙型肝炎病毒核心蛋白及ＮＳ３蛋白在转基因小鼠体内的基因表达及细胞内定位,以及病毒蛋白 直接致细胞病变效应,我们采用ＲＴ－ＰＣＲ方法检测了靶基因ｍＲＮＡ在转基因小鼠各种组织中的表达,用免疫组化方法分析了核心蛋白与ＮＳ３蛋白在转基因小鼠体内的表达及细胞内定位。     

Macronuclear Molecules Encoding Actins in Spirotrichs

Abstract The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared. As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5′ strands in additional actin molecules are purine-rich. This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development. The 50-base segment upstream of the ATG in the 5′ leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements. The 3′ noncoding trailers contain virtually no conserved sequence motifs. With one exception, the 3′ trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon. Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence. Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules. The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.     

Tet-on和Cre/loxP系统双重调控表达丙型肝炎病毒NS3/4A蛋白酶三转基因小鼠的建立

目的:建立Tet-On调控系统和Cre/loxP基因剔除系统双重调控表达丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶三转基因小鼠。方法:选择适龄并经鉴定的在Tet-on系统调控下肝脏特异性表达Cre重组酶的双转基因小鼠Lap/LC-1与在Tet-on系统调控下肝脏特异性表达萤光素酶(Luc)的双转基因小鼠Lap/NS3/4A交配,子代小鼠经PCR检测、筛选基因组中NS3/4A、Lap、LC-1等3个转基因片段均阳性的小鼠。三阳性的NS3/4A/Lap/LC-1小鼠经多西环素(Dox)诱导1周后,以在体生物发光成像系统(BLI)检测报告基因Luc的表达,免疫组化检测小鼠体内Cre重组酶、HCV NS3/4A丝氨酸蛋白酶的表达状况。结果:NS3/4A/Lap/LC-1小鼠经Dox诱导后,BLI结果显示仅在小鼠肝脏部位有强烈的发光信号,表明这些小鼠肝细胞内报告基因Luc特异高效表达;免疫组化结果证实Cre重组酶、NS3/4A蛋白酶仅在经诱导后的小鼠肝细胞中特异性表达。结论:建立了Tet-On调控系统和Cre/loxP基因剔除系统双重调控下表达HCV NS3/4A丝氨酸蛋白酶的三转基因小鼠模型,为进一步研究HCV NS3/4A丝氨酸蛋白酶在HCV感染后与宿主相互作用的机制,以及抗NS3/4A丝氨酸蛋白酶特异性抑制剂的筛选奠定了基础。