A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.     

锯缘青蟹精氨酸激酶基因的克隆与表达

通过分子生物学方法,克隆得到锯缘青蟹（Scylla serrata）精氨酸激酶（Arginine Kinase,AK）开放阅读框基因序列.测序结果显示：该开放阅读框基因的序列长度为1 071 bp,编码356个氨基酸残基;该序列登录Genbank（GQ：851626）,序列比对结果显示,锯缘青蟹精氨酸激酶与凡纳滨对虾（...     

凡纳滨对虾(Litopenaeus vannamei)Hsp70基因PCR扩增及序列分析

目的：为获得凡纳滨对虾的热休克蛋白70（Hsp70）基因并分析其基因序列．方法：根据GenBank中斑节对虾（Penaeus monodon）Hsp70基因的cDNA序列,设计引物,对经高盐法提取的凡纳滨对虾（Litopenaeus vannamei）基因组DNA,采用优化的降落PCR（Touch Downeca）程序,扩增凡纳滨对虾Hsp70基因的全长序列．结果：PCR扩增得到一条长1983bp的目的DNA片段,回收纯化该片段并测定其核酸序列．用DNAman软件分析发现,该核酸序列中不含内含子,编码区全长为1959bp;经BLASTn和BLASTx软件分析发现,该编码区核苷酸序列与斑节对虾、罗氏沼虾（Macrobrachium rosenbergii）的Hsp70基因序列的相似性分别为97％和62．2％．根据核苷酸序列所推导出的Hsp70氨基酸序列,其与斑节对虾、罗氏沼虾的相似性分别为99．9％和92．6％．结论：成功地从凡纳滨对虾基因组DNA中直接扩增出Hsp70基因的全长编码区序列。     

一个在麻疯树根和茎高表达的Kunitz型蛋白酶抑制剂基因JcKTI具有抗虫活性

通过RT-PCR和PCR技术,从麻疯树基因组中克隆得到一个Kunitz型蛋白酶抑制剂基因（JcKTI）的开放阅读框序列。对应开放读码框的基因组序列不含有内含子。该开放阅读框长度为540bp,编码一个含有179个氨基酸残基的成熟多肽,具有典型的Kunitz家族结构特征。组织特异性表达研究显示,JcKTI基因在根和茎中的表达丰度最高,在叶片和种子中表达较低。构建原核表达载体pET32-JcKTI在大肠杆菌BL21中表达,获得纯化的重组蛋白,该蛋白显示出一定的抑制牛胰蛋白酶的活性。将该基因在烟草中过表达,明胶酶法和BAEE法的结果均显示转基因植物的蛋白提取物对胰蛋白酶具有一定抑制作用,进一步的抗虫实验结果表明转基因烟草叶片可使进食后的棉铃虫幼虫生长发育受阻,并减少对叶片的吞食。上述结果暗示JcKTI基因可能在麻疯树根和茎的抗虫应答中扮演着一定的角色。     

用PCR法检测人巨细胞病毒DNA

用聚合酶链式反应（ＰＣＲ）法检测患者尿液中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ．结果表明，自行设计合成的引物位于ＨＣＭＶ基因组早期蛋白基因区，经ＰＣＲ仪扩增一段长４３０ｂｐ的特异序列片段，对正常人基因组ＤＮＡ或其它疱疹病毒ＤＮＡ无交叉反应．此法可检测出少至１０－１６ｇ（０．１ｆｇ）的病毒ＤＮＡ．通过对３５份尿液标本的检测，比较ＰＣＲ法和组织培养法的检测结果完全一致     

Human cytomegalovirus encodes a glycoprotein homologous to MHC class-I antigens

Primary infection with human cytomegalovirus (HCMV) is persistent and widespread, with symptoms that are mostly subclinical but can cause serious illness or death, particularly in immunosuppressed patients. Recently, proteins from HCMV were shown to bind beta 2-microglobulin (beta 2-m) a protein that is normally found associated with the class-I major histocompatibility complex (MHC) antigens, which are essential for self-non-self recognition in the immune response. These findings led to the proposal that the virus may use beta 2-m binding as an infection mechanism. Here we present evidence from DNA sequence analysis that HCMV encodes a molecule similar to the MHC class-I antigens of higher eucaryotes, and propose that this protein is responsible for the observed beta 2-m binding. The deduced amino-acid sequence of the HCMV class-I-like protein reveals conservation of typical features of class-I structure, but we predict that the gene is not spliced, in contrast to the cellular genes.     

生物信息学技术克隆并分析新基因STRF7

为进一步研究信号转导相关的新基因片段BE644250,采用生物信息学方法克隆基全长cDNA,并分析了其ORF,电子表达谱,染色体定位等,之后对全长序列进行了实验验证。电子延伸（contig)获得了729bp的延伸产物,含一个典型的74aa的ORF,命名为STRF7。与已知蛋白无明显同源性,部分地相似于人的源框蛋白CDX－4和酵母的转录调节子ADR6,属一新发现的基因;RT－PCR从IL－6刺激后的U937中克隆了STRF7基因,基序列与电子延伸结果安全一致,进一步的分析显示STRF7在多种组织中表达并定位于第6号染色体上,上述结果显示,STRF7是一个新基因,编码含74aa的蛋白,并且是一个潜在的转录因子。     

Complete nucleotide sequence of the AIDS virus, HTLV-III

The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3'-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.     

哺乳动物基因非翻译区开放阅读框架的分析

在所分析的5'UTR序列中约19%的序列在阅读框1下含有AUG,约22%和24%的序列分别在阅读框 2 和阅读框3下含有 AUG,而在 3'UTR序列当中,无论是在哪个阅读框下,约71%含有AUG.这些AUG绝大多数都是以小ORF的形式出现的;分析小ORF序列以及与它们相应的IC序列(阅读框间序列)长度,发现虽然3'UTR序列远远长于5'UTR序列,但是它们所包含的小ORF长度几乎没有差距,只是平均来说每一条3'UTR序列所包含的小ORF数量明显多于每一条5'UTR序列所包含的小ORF数量,而且dIC(下游阅读框间序列)比uIC(上游阅读框间序列)长很多,平均长50个碱基;第二类IC序列(UTR区相邻的两个阅读框之间的序列)的平均长度比其它两类IC序列(UTR区最后一个阅读框之后的序列)的平均长度小,而且又含有相对较多的终止密码;比较uORF序列与dORF序列密码子使用偏好的CAI值发现,尽管相差不大,但是无论在哪个阅读框下,uORF序列的CAI值都显著高于dORF序列的CAI值.     

Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immune-compromised individuals, including organ transplant recipients and individuals with AIDS. It is also a leading cause of virus-associated birth defects and is associated with atherosclerosis and coronary restenosis. HCMV initiates infection and intracellular signalling by binding to its cognate cellular receptors and by activating several signalling pathways including those mediated by mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, interferons, and G proteins. But a cellular receptor responsible for viral entry and HCMV-induced signalling has yet to be identified. Here we show that HCMV infects cells by interacting with epidermal growth factor receptor (EGFR) and inducing signalling. Transfecting EGFR-negative cells with an EGFR complementary DNA renders non-susceptible cells susceptible to HCMV. Ligand displacement and crosslinking analyses show that HCMV interacts with EGFR through gB, its principal envelope glycoprotein. gB preferentially binds EGFR and EGFR-ErbB3 oligomeric molecules in Chinese hamster ovary cells transfected with erbB family cDNAs. Taken together, these data indicate that EGFR is a necessary component for HCMV-triggered signalling and viral entry.     

A product of the prune locus of Drosophila is similar to mammalian GTPase-activating protein.

The X-linked prune (pn) eye-colour mutation of Drosophila melanogaster has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation. Although awdK-pn flies have no apparent phenotype on their own, pn awdK-pn double mutants die as second or third larval instars. The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila. Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product. Here we report the nucleotide sequence of TcD37 and the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras (ref. 6), which are plasma membrane-bound proteins involved in the regulation of cell proliferation and differentiation. These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast. We propose that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function.     

A complex between the MHC class I homologue encoded by human cytomegalovirus and beta 2 microglobulin

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that persists in the host and can cause severe disease in the immunocompromised individual or in the fetus. Analysis of the nucleotide sequence of the virus genome has revealed the presence of an open reading frame whose predicted translation product has homology with the heavy chain of the major histocompatibility complex (MHC) class I molecule of higher eukaryotes, and the observed sequence homology was given additional significance by the independent observation that HCMV virions can bind beta 2 microglobulin (beta 2m), the light chain of the MHC class I molecule. We expressed both the HCMV class I homologue and the human beta 2m gene in recombinant vaccinia viruses. We show that the coexpressed gene products associate, that the transport of beta 2m to the cell surface is dependent on coexpression of the class I homologue and that the viral gene product is therefore functionally related to its cellular counterpart. We observe also that, in HCMV-infected cells, no synthesis of mature cellular class I molecules occurs, while messenger RNA levels remain unaltered, and we speculate that one function of the viral homologue may be to sequester beta 2m, thus preventing the maturation of cellular class I molecules and rendering the infected cell unrecognizable by cytotoxic T cells.     

大熊猫（Ailuropoda melanoleuca）RPS24基因cDNA克隆及序列分析

本研究运用RT—PCR技术,首次从大熊猫（Ailuropoda melanoleuca）的肌肉组织总RNA中成功克隆了RPS 24（Ribosomal Protein S24）基因的表达序列,并对其进行了测序及初步分析．结果表明：大熊猫RPS24基因的表达序列全长为431bp,开放阅读框（ORF）为399bp,编码132个氨基酸的蛋白质,该蛋白的分子量为15．3251kD,pI为10．92,含有7种类型共14个功能位点：即1个N-糖基化位点、2个cAMP和cGMP依赖性蛋白激酶磷酸化位点、6个蛋白激酶C磷酸化位点、1个酪蛋白激酶Ⅱ磷酸化位点、3个N-酰基化位点、1个酰胺化位点及1个RPS 24e signature．进一步分析发现,大熊猫RP524基因与已报道的人、牛、苏门答腊猩猩、褐家鼠和小家鼠5个哺乳动物物种的表达序列及其编码的氨基酸序列具有很高的相似性：编码序列同源性分别为94．1％、92．4％、94．7％、89．9％和90．4％;与人RPS24蛋白的氨基酸序列同源性为98．48％,其余均为99．24％．     

Molecular cloning and expression analysis of a novel human cDNA fragment encoding a putative Ser/Thr protein kinase

A novel full-length cDNA encoding a putative serine/threonine kinase has been isolated from a human testis cDNA library. A nucleotide sequence of 1225 bp length has been determined containing an open reading frame of 1 044 nucleotides (encoding 348 amino acids). In view of its degree of homology to members of the Ser/Thr protein kinase family and the closest relationship toMus musculus STK-1, the predicted product was designated by the name of HUMSTK-1. Its mRNA is present in large amounts in thymus, and small amounts in testis, small intestine and colon.     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

棉花黄萎病菌VdSgel基因的克隆与序列分析

为了探索棉花黄萎病菌（Verticillium dahliae Kleb）是否存在转录调节因子VdSgel，本研究以新疆棉花黄萎病菌强致病力菌株V592为材料，通过设计引物，对棉花黄萎病菌进行了VdSgel基因的克隆和测序。获得VdSgel基因全序列为1533bp，不含内含子，只编码1个含510个氨基酸的蛋白（VdSgel）。VdSgel的N端含有真菌特异的TOS9结构域（COG5037）和Gti1—Pac2基因家族结构域（Pfam09729）。此外N端还含有一个推测的蛋白激酶磷酸化位点（^66KRWTDG^71）及一个核定位信号（^94PGEKKR^100）。系统进化分析结果表明，VdSgel与番茄枯萎菌、荚膜组织胞浆菌、小麦赤霉菌和葡萄孢菌的同源调节因子进化关系较近。本研究为进一步研究VdSgel的功能奠定了基础。     

马铃薯天冬氨酸蛋白酶抑制剂基因克隆和结构分析

以马铃薯基因组ＤＮＡ为模板并基于天冬氨酸蛋白酶抑制剂基因的ｃＤＮＡ序列所设计的两个引物，通过ＰＣＲ扩增得到６６６ｂｐ的天冬氨酸蛋白酶抑制剂基因编码区，并将此片段克隆到ｐＵＣ１８的ＳｍａＩ位点．序列分析结果表明该基因除去末端一终止密码子外其可译框架编码２２１个氨基酸残基，前导肽包括３２个氨基酸残基，故该抑制剂的成熟蛋白由１８９个氨基酸组成，第６７位的精氨酸为抑制胰蛋白酶的活性中心．与ｃＤＮＡ相比核苷酸的同源性为９４．３％，氨基酸的同源性为９０％．基因ＤＮＡ无内含子．     

A dynamin-like protein encoded by the yeast sporulation gene SPO15.

The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae. Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles. Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body. Thus cells are unable to form a bipolar spindle. Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids. This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin. The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules. SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.