A Putative Receptor Protein Kinase Gene in Ipomoea trifida

We have characterized a cDNA clone, IRK1, for a putative receptorkinase from a stigma cDNA library of Ipomoea trifida. IRK1 proteincontains an extracellular receptor-like domain and the consensussequences diagnostic of serine/threonine protein kinase. Boththe pattern of gene expression and the results of RFLP analysisindicate that the IRK1 gene is not primarily involved in theself-incompatibility system of Ipomoea. (Received October 16, 1995; Accepted April 10, 1996)     

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.     

一个推测的拟南芥漆酶基因在巴斯德毕赤酵母中的表达

At5g01040基因是一个推测的拟南芥漆酶基因。根据其编码序列设计引物,利用RT—PCR方法扩增出1755bp的片段。测序结果表明,该片段存在3个突变位点,其中一个突变位点改变了所编码的氨基酸。将该片段克隆到表达载体pPICZaB上,电击转化毕赤酵母。经筛选获得80个候选重组菌株,其中18个菌株的培养液中存在漆酶活性,说明所克隆的基因在毕赤酵母中实现了分泌表达,证实了At5g01040编码的蛋白具有漆酶活性。     

Gene Mapping with Recombinant Inbreds in Maize

Recombinant inbred lines of maize have been developed for the rapid mapping of molecular probes to chromosomal location. Two recombinant inbred families have been constructed from F(2) populations of T232 X CM37 and CO159 X Tx303. A genetic map based largely on isozymes and restriction fragment length polymorphisms has been produced that covers virtually the entire maize genome. In order to map a new gene, an investigator has only to determine its allelic distribution among the recombinant inbred lines and then compare it by computer with the distributions of all previously mapped loci. The availability of the recombinant inbreds and the associated data base constitute an efficient means of mapping new molecular markers in maize.     

SARS冠状病毒S基因序列克隆的构建

构建SARS冠状病毒S基因序列克隆p-SARS-S,作为自行设计、建立的RT-PCR检测方法的阳性对照,并为该基因的表达奠定基础.设计合成了位于病毒基因21 504～22 136位的长633 bp的序列片断作为模板进行PCR扩增;将PCR产物与T-Easy载体连接,经过转化提取重组质粒DNA.对构建的阳性克隆进行PCR、酶切、测序鉴定.对经EcoRⅠ酶切鉴定为阳性的重组表达质粒测序,结果显示插入片段大小、方向、碱基匹配与预期一致.构建的阳性对照克隆p-SARS-S有助于建立严格的SARS病毒基因室内质控措施.     

SARS冠状病毒M蛋白的生物信息学研究

针对GenBank上发布的来自不同国家地区的39条SARSCoV推测M蛋白,采用生物信息学软件分析其核酸和氨基酸序列,获得其分子生物学特征,确定突变位点,预测功能结构区、Motif及抗原决定簇,比较基因突变对这些功能结构的影响.结果表明:在39个病毒株M蛋白的666 bp中,共有18个病毒株在7个位点上发生了25次变异.在M蛋白序列上预测获得3个跨膜螺旋序列和一个可能的信号肽序列.氨基酸序列的变异主要发生在其跨膜和胞外区域,胞内区域相对较少.预测发现12个Motif和7个抗原决定簇.提示突变对M蛋白的结构功能区的影响不大,也未造成M蛋白的Motif的数量和构成发生改变.对抗原决定簇的影响也主要体现在序列成分构成的改变上,在设计疫苗时,应考虑由其导致的抗原特性改变.     

重组SARS冠状病毒刺突蛋白的表达和分离纯化

SARS冠状病毒的感染能引发人的严重急性呼吸综合征。根据对其他种类冠状病毒的研究结果 ,刺突(spike)蛋白 (S蛋白 )是病毒的主要表面抗原 ,重组S蛋白可用于临床诊治 ,疫苗制备和结构生物学研究。SARS病毒S蛋白基因被分段和完整地克隆到不同的细菌表达载体进行了表达。通过宿主菌的选择和条件的优化 ,其中75 1～ 192 5bp、2 0 0 5～ 3410bp、1～ 192 5bp、32～ 36 5 9bp片段及全长 1～ 376 8bpDNA都在大肠杆菌中实现了高效表达 ,表达量分别占菌体蛋白质的 35 %、34%、2 4 %、17%和 5 % ,并经亲和层析得到了部分纯化。纯化后的蛋白质将用于诊断试剂和结构生物学研究。     

一个受褐飞虱取食诱导的水稻GST类似蛋白基因的克隆

BpHi006AcDNA长度为1943bp,具有一个795bp组成的完整的读码框,其表达受褐飞虱取食的诱导。BpHi006A蛋白含有谷胱苷肽S转移酶(glutathione Stransferase)的N末端结构域和C末端结构域,为谷胱苷肽S转移酶超家族的成员。BpHi006A蛋白与拟南芥四氯氢醌还原性脱卤素酶相关蛋白有61％的一致性,序列分析表明这两种蛋白质构成一类新的植物GST。     

乙型肝炎病毒前S基因区缺失突变发生机制的探讨

检测慢性乙型肝炎病毒(HBV)携带者和患者外周血内HBV前S区基因缺失突变的分子结构特点,探讨其发生机理.用聚合酶链反应方法从慢性乙肝患者和携带者血清中扩增出前S区基因片段,克隆、测序,分析缺失发生的结构特点,从而推测这些前S区基因缺失突变的产生机制.从262例慢性乙肝患者和103例无症状HBV携带者体内扩增出前S区片段,共在30例患者和携带者中检测出多种前S区基因缺失突变,主要集中于前S1区的3′端和前S2区的5′端.其中有9例患者和携带者体内存在完全一样的nt3019～nt3201 183bp的缺失突变,该缺失突变符合真核细胞mRNA剪接机制,在此位置上各基因型的序列高度保守.同时有另外两种缺失突变,即nt3019～nt3147 129bp缺失、nt3019～nt3109 91bp缺失也符合该剪接机制.有23种缺失突变部分于重复序列之间,符合逆转录过程中的模板转换机制所导致的缺失.根据前基因组RNA预测出二级结构,仅部分缺失突变在RNA二级结构中对应于局部的结构.此结果表明:HBV在外界因素mRNA的剪接机制和内在因素聚合酶蛋白的功能特点的共同作用下,产生各种突变,不同的机制将导致不同类型的缺失突变.除真核细胞mRNA剪接机制外,逆转录过程中的模板转换是主要机制之一.     

人细胞骨架调节蛋白基因NELIN cDNA的克隆及特征分析

为寻找和研究心血管系统有关的重要功能基因及表达模式,构建了正常成人心脏和主动脉cDNA文库,并在大规模表达序列标签（ESTs）测定和筛选新的cDNAs全长的基础上,筛选出一个新的基因（GenBank登记号AF114264）。该基因的cDNA全长为2736bp,含有一个1344bp的开放读码框,由于其推测的氨基酸序列与鼠源微管连接蛋白（nexilin）具有很高同源性,所以暂将其命名为NELIN（nexilin-like protein)。Northern印迹和RT－PCR结果表明,该基因的心脏、骨骼肌、动脉和静脉中表达,且该表达有一定的时空特异性,查询GeneMap‘99,该基因定位在梁色体1p31－1p32。结构域分析表明,NELIN很可能参与调节粘着斑和张力纤维形成,并参与粘着斑的信号转导。     

新分离呼吸道肠道病毒生物学特性和S4片段的序列测定

新分离呼吸道肠道病毒(简称呼肠病毒)经过空斑分纯后进行生物学和部分基因序列的鉴定.首先通过BYD株(由端青从北京于某患者标本中分离到的第一株呼肠病毒)对4种传代细胞的敏感性试验,选出L929和LLC-MK2敏感细胞,然后用LLC-MK2细胞进行理化性质试验,血凝试验和形态学鉴定.结果显示该病毒为耐乙醚、不耐酸、不耐热的RNA病毒;对人O型红细胞能产生凝集,滴度达132;电镜下可查见细胞胞浆内有球形、直径为70nm～80nm、双层衣壳的病毒颗粒.提取BYD株RNA并进行S4片段序列分析,结果显示该片段的氨基酸序列与已知呼肠病毒1～3型标准株的同源性分别为95%、90%和95%.以上这些生物学以及S4片断的序列分析结果表明新分离BYD株病毒符合呼肠病毒科的特性.     

新分离呼吸道肠道病毒生物学特性和S4片段的序列测定

新分离呼吸道肠道病毒(简称呼肠病毒)经过空斑分纯后进行生物学和部分基因序列的鉴定。首先通过BYD株(由端青从北京于某患者标本中分离到的第一株呼肠病毒)对4种传代细胞的敏感性试验,选出L929和LLC-MK2敏感细胞,然后用LLC-MK2细胞进行理化性质试验,血凝试验和形态学鉴定。结果显示该病毒为耐乙醚、不耐酸、不耐热的RNA病毒;对人O型红细胞能产生凝集,滴度达1:32;电镜下可查见细胞胞浆内有球形、直径为70nm～80nm、双层衣壳的病毒颗粒。提取BYD株RNA并进行S4片段序列分析,结果显示该片段的氨基酸序列与已知呼肠病毒1～3型标准株的同源性分别为95%、90%和95%。以上这些生物学以及S4片断的序列分析结果表明:新分离BYD株病毒符合呼肠病毒科的特性。     

dad-1, A Putative Programmed Cell Death Suppressor Gene in Rice

The human dad-1 cDNA homolog was isolated from rice plants.The amino acid sequence of the predicted protein product iswell conserved in both animals and plants. This rice dad-1 homologcan rescue the temperature-sensitive dad-1 mutants of hamstercells from apoptotic death, suggesting that the rice dad-1 homologalso functions as a suppressor for programmed cell death. (Received December 24, 1997; Accepted January 27, 1997)     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

Isolation and Characterization of a Putative Class E Gene from Taihangia rupestris

Studies In model plants showed that SEPALLATA （SEP） genes are required for the Identification of floral organs and the determination of floral meristems In Arabidopsis. In this paper a SEP homolog, TrSEP3, was Isolated from a China-specific species, Taihangla rupestrisi Yü et LI. Phylogenetlc analysis showed that the gene belongs to the SEP3-clade of SEP （previous AGL2） subfamily. In situ hybridization was used to reveal the potential functional specification, and the results showed that TrSEP3 expression was first observed in floral meristems and then confined to the floral primordla of the three inner whorls. In the matured flower, TrSEP3 was strongly expressed In the tips of pistils and weak In stamens and petals. The evolution force analysis shows that TrSEP3 might undergo a relaxed negative selection. These results suggested that TrSEP3 may not only function In determining the identity of floral merlstems and the primordia of three inner whorls, but also function In matured reproductive organs.     

Identification of a Structural Gene Encoding a Metallothionein-like Domain that Includes a Putative Regulator Protein for Streptomyces Protease Gene Expression

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.