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.     

Characterization of a calmodulin binding protein kinase from<Emphasis Type="Italic">Arabidopsis thalian</Emphasis>

A full-length calmodulin binding protein kinase cDNA ,AtCBK1 ,from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE-Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues,The phylogenetic analyses revenl that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup,indicating that they may come from different ancestors,The result suggests that AtCBK1 encoldes a CaM-binding serine/threonine protein kinase.     

Molecular cloning, identification and characteristics of NYD-SP9: Gene coding protein kinase presumably involved in spermatogenesis

Using cDNA microarray hybridization from a human testicular cDNA library, one gene exhibiting ten-fold difference at expression level between adult and embryo human testes was cloned and named NYD-SP9, which was believed to be involved in spermatogenesis. Southern blot hybridization results showed that NYD-SP9 expressed highly in testis but low in ovary. Protein motif analysis of this cDNA sequence revealed a cluster of phosphorylation sites, indicating its potential involvement in signal pathways during spermatogenesis. Furthermore, one transmembrane helix was predicted in N-terminal region, indicating that putative NYD-SP6 may be served as a transmembrane protein. The proximity of these potential phosphorylation sites to each other indicates that there may be interaction among these sites to regulate spermatogenesis. These findings suggested that protein kinase NYD-SP9 might play a role in male germ cell differentiation.     

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

猪Akt1、Akt2基因克隆与组织表达图谱分析

为了得到长白猪蛋白激酶Akt1和Akt2基因序列并分析其表达模式,本研究使用RT-PCR方法,首先克隆了蛋白激酶Akt1和Akt2的cDNA.序列分析显示:长白猪Akt1基因的cDNA全长1461bp,编码具480个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠同源性达到97%以上.长白猪Akt2基因cDNA全长为1505bp,编码具481个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠的同源性高达97%以上.SMART分析表明,猪Akt1和Akt2蛋白均包含了与PI-3K结合的PH结构域及2个具有丝氨酸/苏氨酸激酶催化活性的S_TKc结构域.RT-PCR检测结果显示:Akt1mRNA在垂体、心脏、肝脏、脾脏、肌肉组织中高表达,在大脑、小脑、肾脏表达丰度较低.Akt2则在小脑、垂体、心脏、肝脏、脾脏和肌肉组织中高表达,而在大脑和肾脏中表达丰度较低.     

Isolation and cloning of a novel cDNA LDBl 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 Ldb1. 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 Ldb1, Xenopus Xldb1 and Drosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been named LDB1 (LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts of LDB1 in heart, brain and lung were considerably higher than those in other tissues.     

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.     

Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of<Emphasis Type="Italic">Artemisia annua</Emphasis> L.

A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1(FPS,EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has heen cloned from its cDNA library .Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD.Deduced aa sequence of the cDNA was similar to FPS from other plants,yeast and mammals,containing 5 conserved domains found in both prenyl trans-ferase and polyprenyl synthase,The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro.The enzyme was purified by ion exchange chromatography and its kinetics was measured ,These results would further promote the molecular regulation of artemisnin biosynthesis.     

Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

A new MAPK gene, ZmSIMK1 （Zea mays L. salt-induced mitogen-activated protein kinase 1）, is isolatod from a maize eDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of eneoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 eDNA into pET-42a（＋）, and transformed into E. coli strain BL21（DE3）. A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1 mmol/L IPTG.     

Molecular cloning and expression of a rat V1a arginine vasopressin receptor.

The neurohypophyseal hormone arginine vasopressin has diverse actions, including the inhibition of diuresis, contraction of smooth muscle, stimulation of liver glycogenolysis and modulation of adrenocorticotropic hormone release from the pituitary. Arginine vasopressin receptors are G protein-coupled and have been divided into at least three types; the V1a (vascular/hepatic) and V1b (anterior pituitary) receptors which act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+, and the V2 (kidney) receptor which is coupled to adenylate cyclase. We report here the cloning of a complementary DNA encoding the hepatic V1a arginine vasopressin receptor. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA corresponding to the cDNA is distributed in rat tissues known to contain V1a receptors.     

Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.     

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.