Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus.

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.     

A Met-to-Val mutation in the skeletal muscle Na+ channel alpha-subunit in hyperkalaemic periodic paralysis.

HYPERKALAEMIC periodic paralysis (HYPP) is an autosomal dominant disease that results in episodic electrical inexcitability and paralysis of skeletal muscle. Electrophysiological data indicate that tetrodotoxin-sensitive sodium channels from muscle cells of HYPP-affected individuals show abnormal inactivation. Genetic analysis of nine HYPP families has shown tight linkage between the adult skeletal muscle sodium channel alpha-subunit gene on chromosome 17q and the disease (lod score, z = 24; recombination frequency 0 = 0), strongly suggesting that mutations of the alpha-subunit gene cause HYPP. We sequenced the alpha-subunit coding region isolated from muscle biopsies from affected (familial HYPP) and control individuals by cross-species polymerase chain reaction-mediated complementary DNA cloning. We have identified an A----G substitution in the patient's messenger RNA that causes a Met----Val change in a highly conserved region of the alpha-subunit, predicted to be in a transmembrane domain. This same change was found in a sporadic case of HYPP as a new mutation. We have therefore discovered a voltage-gated channel mutation responsible for a human genetic disease.     

模板甲基化水平对鼠肝RNA聚合酶体外转录活性的影响

利用３Ｈ－ＵＴＰ同位素参入法，研究了大鼠肝细胞ＲＮＡ聚合酶在核内外的转录活性，结果表明：在细胞核内的转录活性明显低于在无核抽提物中的转录活性。利用自身的ＤＮＡ模板，优于利用外加的ＤＮＡ模板。并比较了不同ＤＮＡ甲基化水平的模板对ＲＮＡ聚合酶体外转录活性的影响，发现模板的甲基化水平与其体外转录水平呈反相关。     

广西巴马小型猪肌肉生长抑制素(Myostatin)cDNA克隆和序列分析

目的通过RT-PCR扩增广西巴马小型猪Myostatin cDNA序列,经过连接、转化、克隆测序后,与NCBI中发表的猪Myostatin序列进行同源性比较,分析广西巴马小型猪Myostatin的cDNA序列结构特点及与其他物种间的聚类关系,为日后构建Myostatin高效表达载体及进一步研究Myostatin对广西巴马小型猪肌肉生长的影响奠定基础。方法以广西巴马小型猪的肌肉组织总RNA为模板,应用逆转录-聚合酶链式反应(RT-PCR),利用合成的特异性引物,扩增得到巴马小型猪肌肉生长抑制素(Myostatin)cDNA的全序列。该扩增片段经纯化后,连接到pMD18-T载体上扩增,经一系列鉴定后,测序。结果本实验克隆到巴马小型猪Myostatin基因cDNA序列长度为1128 bp,与GenBank报道的猪Myostatin基因cDNA序列同源性高达99.7%,与人、奶牛、家鼠等物种的cDNA序列间同源性可达到92.3%以上,证明Myostatin基因具有较高的保守性。同时发现广西巴马小型Myostatin基因cDNA序列有三处存在碱基突变,两个为同义突变,一个为错义突变。