Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.     

Cloning and identification of measles virus receptor gene from marmoset cells

The measles virus (MV) strains with mutated hemagglutinin gene (ha) lost the capacity to infect its sensitive host cells (Vero cells), but it may infect the marmoset B-lymphoblastoid cell line B95a. From above, we can presume that there is a novel cellular receptor for those measles virus strains on B95a cell s. Using the yeast two-hybrid system, we screened and cloned a novel gene--bip (B-lympho- blastoid interaction protein of marmoset) from B95a cell cDNA library, which encoded a protein interacting with measles virus hemagglutinin protein (Ha). The bip cDNA was 1540 base pairs in length and contained a unique open rea ding frame (ORF) of 1011 base pairs encoding a transmembrane protein of 337 amino acid residues. The primary structure of amino acids residue is predicted that the Bip comprised a hydrophobic transmembrane domain and a hydrophobic leader region. The researches about the deletion mutants showed that the deletion of tran smembrane domain in Bip did not affect the interaction between Bip and Ha protei ns. Expression of bip in measles virus non-permissive cell line--CHO (Chinese hamster ovary) cells was performed to prove that CHO/Bip can be infected by meas les virus and then turned to the MV permissive cells. We concluded that the bip gene is a novel measles virus receptor gene in marmoset B-lymphoblastoid cells.     

Cloning and identification of a novel binding protein of CD2 cytoplasmic domain

In order to understand why CD2 has a dual action of transduction of activation or apoptosis signals in T cells under different experimental conditions, we employed a yeast two-hybrid system to look for a binding protein of the cytoplasmic domain of CD2 which may be involved in this issue. A human T cell cDNA library was screened by a cDNA encoding the cytoplasmic domain of CD2 (Thr211-Gln336). The specificity of protein-protein interaction was verified by co-immunoprecipitation. The binding protein obtained, designated CD2cBP, was found to be homologous tov-fos transformation effector protein (Fte-1). As Fte-1 plays a role in cell transformation, growth, protein synthesis and protein-import into mitochondria, this result suggests that CD2cBP may be putatively involved in CD2-mediated signaling.     

嗜热真菌Thermomyces langinosus编码丝/苏氨酸蛋白激酶部分cDNA的克隆及序列分析

根据几种丝状真菌保守的氨基酸序列,设计了一组简并性的引物,从嗜热真菌Thermomyceslanginosus中提取总RNA,利用RT-PCR的方法,我们得到了一个1243bp的cDNA的片段。片段回收纯化后连接到pGEM-Teasy载体上,PCR扩增重组质粒证实这个长约1200bp片段已经插到载体上。序列分析发现和裂殖酵母的蛋白激酶dis1非常相似,比较两者3'氨基酸序列发现同源性达28%以上。其推断的氨基酸序列上包含有6个丝/苏氨酸蛋白激酶的保守亚区。这些都表明它编码的基因可能是一个新的丝/苏氨酸蛋白激酶。     

Influence of PrP 106―126 on expression of laminin and fibronectin in astrocyte

Astrogliosis is a hallmark of prion disease, but the metabolic alterations of astrocytes remain poorly documented. A synthetic peptide corresponding to amino acid 106-126 of the human prion protein （PrP） has been shown to be toxic to neurons. In this study, the effects of PrP 106-126 on astrocytes were investigated in vitro. The proliferation of astrocytes was significantly （P 〈 0.05） increased when grown in media conditioned with PrP 106-126 （80 μmol/L） from microglia. The expression of laminin （LN） and fibronectin （FN） was examined at both mRNA and protein levels. The results showed that exposure of astrocytes to PrP 106-126 enhanced the expression of LN and FN. The increase of FN in astrocyte cultures required cytokines previously released by activated microglia. This study reveals the expression of LN and FN affected by PrP106-126.     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

Study on the interaction between Jak3 and IL-2R γ using the yeast two-hybrid system

Both interleukin-2 (IL-2) receptor γ subunit and non-receptor tyrosine kinase Jak3 play important roles in IL-2 physiological functions. Jak3 has been known to bind IL-2Rγ and the interaction is very important for IL-2 signaling. In order to find the domains directly involved in the interaction between IL-2Rγ and Jak3, various deletion mutants have been constructed and their interaction has been studied using the yeast two-hybrid system. Results show that the JH3-JH7 region of Jak3 N-terminal can bind to intracellular domain of IL-2Ry directly and the intact structure of JH3-JH7 region is necessary for this combination. In addition, the region from 305 to 317 amino acid residues near the C-terminal of IL-2Ry plays a critical role in this interaction .     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Isolation and cloning of a novel cDNALDB1 encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

hASB-8相互作用蛋白的初步研究

hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因．其编码蛋白属于人ASB蛋白家族中的一个成员,与小鼠中的ASB-8蛋白同源性达96％．保守结构域分析显示hASB-8在N端包含4个Ankyrin repeats,在C端包含了一个SOCS box．利用酵母双杂交技术,筛选了人的胎盘(Placenta)cDNA文库,获得了与KASB-8相互作用的2个蛋白,Elongin C和CDK4 binding protein;并在二倍体酵母体内进行了验证．这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用,并与肿瘤细胞靶蛋白转录调节有关．     

Identification and characterization of a salt tolerance-responsive gene( AtGRP9) of Arabidopsis

Soil salinity is one of the important limiting factors for plant growth and development. A cDNA clone encoding a glycine-rich protein (designated AtGRP9) was identified from Arabidopsis by functional expression of the plant cDNA library in the fission yeast S. pombe. Yeast cells overexpressing AtGRP9 displayed significantly enhanced salt tolerance. Northern analysis showed that expression of AtGRP9 in Arabidopsis was induced by NaCl and plant hormone abscisic acid (ABA). These results suggest that AtGRP9 may be involved in the salt stress response in Arabidopsis.     

PAS domain of the deduced Org35 protein mediates the interaction with NifA

PAS domains are sensory input domains and pro- tein-protein interaction sites that have been identified recently in a family of sensory proteins from all king-doms of life[1,2]. A variety of environmental stimuli such as light, oxygen, redox potential, an…     

Interaction of Stathmin-Like 2 Protein with the APP Intracellular Domain

Amyloid precursor protein （APP） plays a critical role in the formation of Alzheimer＇s disease （AD）. The intracellular domain APP （AID） was suggested to cause neurotoxicity in the nucleus. To investigate the functions AID, yeast two-hybrid screening was performed to identify the proteins which interact with AID. The human brain cDNA library was screened using pGBKT7-AID as a bait, and several positive clones were identified. One of them encodes a part of the human stathmin-like 2 protein （STMN2）. The interaction between STMN2 and APP implies that STMN2 may have an important function in APP-mediated AD formation.     

平颏海蛇CRVP基因全长cDNA片段的克隆和序列分析

通过对平颏海蛇毒腺cDNA文库的随机测序和PCR筛选,克隆得到两种分别长为1309和1307bp的编码半胱氨酸丰富毒蛋白（cysteine-rich venom protein,CRVP）的全长cDNA序列。这两种crvp基因同源性为97％,分别编码238和199个氨基酸残基,其中包括一段相同的由19个氨基酸残基组成的信号肽。氨基酸序列分析表明,这两种CRVP蛋白与蜥蜴helothermine毒素蛋白以及台湾饭铲倩CRVP毒蛋白高度同源,而且具有半胱氨酸丰富分泌蛋白（cysteine-rich secretory protein,CRISP)家族的典型结构特征。     

抗冻蛋白基因定向突变及其抗冻关系的研究

为探讨抗冻蛋白的抗冻机制,根据抗冻活力较高的肝型抗冻蛋白的氨基酸组成,改造皮肤型抗冻蛋白基因,使皮肤型抗冻蛋白基因的10位氨基酸－丙氨酸（A）改造成天冬氨酸（D）,方法：合成一段含有突变位点的DNA片段,用其取代野生型DNA上相对应的片段,DNA测序法筛选阳性克隆并表达该蛋白,Western-blot法、氨基酸组成分析法、及质谱法鉴定表达蛋白,测定突变后抗冻蛋白的的活力并与野生型比较,以期揭示抗冻蛋白的抗冻机制。