Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

人类新基因cegl cDNA的克隆及表达研究

CLECT and EGF-like domain contained Gene 1(cegl)基因是用电子克隆的方法获得的人类新基因。该基因定位在人类的第14号染色体上,是一个单一外显子的基因。cegl基因的cDNA长度为2050bp,通过生物信息学方法预测它包含一个1340bp的完整阅读框架,编码一个490个氨基酸的蛋白,含有CLECT、EGF-like结构域各一个。以cegl基因全长编码区为探针的整体原位杂交结果显示该基因的小鼠和鸡的同源基因在各自早期胚胎头部中特异表达,并且在不同时期胚胎神经系统增殖迅速的部位中有大量的表达。RT-PCR结果显示该同源基因在小鼠成体各组织中广泛分布。这提示cegl基因可能与头部生长发育有密切关系,并且对维持成体各组织的正常功能起到重要的作用。对cegl基因在胚胎发育的时间和空间表达模式的研究将有助于进一步深入地揭示它在人脑的正常生长发育中的作用。     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Cloning, expression and characterization of a novel human REPS1 gene.

Ral is a member of the small GTPase-binding protein (G protein) family, and plays an important role in the Ras-RalGDS signal transduction pathway. A series of recent findings reveal several important downstream target proteins of Ral, such as RalBP1, Reps1, and others. Here we report another binding partner for RalBP1, which we have isolated from the human fetal brain library. The human REPS1 protein shares 83% amino acid identity with the mouse Reps1 protein. Northern blot analysis shows that the REPS1 is expressed in a variety of tissues, with the strongest expression in the heart and testis.     

Cloning and pharmacological characterization of a novel gene encoding human nucleoside transporter 1 (hNT1) from a human breast cancer cDNA library

AimsWe isolated a novel gene encoding human nucleoside transporter 1 (hNT1), from a human breast cancer cDNA library.Main methodsA nondirectional cDNA library was screened by an EST clone (GenBank?/EMBL/DDBJ: BU944345). A Xenopus laevis oocyte expression system was used for functional characterization. Membrane localization in the human breast was determined by immunohistochemistry.Key findingsIsolated hNT1 cDNA consisted of 246 base pairs that encoded an 82-amino acid protein. By RT-PCR analysis, hNT1 mRNA was strongly detected in the breast cancer tissues. When expressed in X. oocytes, hNT1 mediated the high affinity transport of [³H]5-fluorouracil (5-FU) with a K_m value of 69.2 ± 24.5 nM in time- and pH-dependent, and Na⁺-independent manners. A cis-inhibition experiment revealed that hNT1 mediated transport of [³H]5-FU is strongly inhibited by various nucleosides such as pyrimidine, uracil, uridine, guanosine, inosine, thymidine, adenosine, cytidine and purine suggesting that hNT1 may be involved in the trans epithelial transport of these endogenous substrates. Immunohistochemical analysis revealed that the hNT1 protein is localized in the lactiferous duct epithelium.SignificanceOur present results indicate that a newly isolated cDNA clone, hNT1, is a key molecule for the breast handling of 5-FU in humans.     

Cloning and characterization of a novel human RNA binding protein gene PNO1.

Present work reported the cloning and characterization of a human novel RNA binding gene Partner of NOB1 (PNO1), with a length of 1637bp and a putative open reading frame of 759 bp, isolated from human kidney. It is composed of seven exons and is localized on chromosome 2p14. Western blot showed that the molecular weight of PNO1 is about 35kDa. RT-PCR results in 16 human tissues indicated that PNO1 is expressed mainly in liver, lung, spleen and kidney, slightly in thymus, testis, ovary, respectively, but not in heart, brain, skeletal muscle, placenta, pancreas, prostate, small intestine, colon and peripheral blood leukocytes. GFP fusion expression in mammalian cells exhibited its localization in the nucleus, especially in nucleoli. Subcellular localization of thirteen GFP fusion PNO1 deletion proteins showed that the region of 92-230 aa is solely responsible for its nucleolar retention, and KH domain alone is not sufficient for nucleolar retention. The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.     

Cloning and characterization of a novel trypsin inhibitor (BTIomega1) gene from Fagopyrum esculentum.

Based on the amino acid information of trypsin inhibitor of buckwheat (Fagopyrum Esculentum Moench), degenerated primers were designed and a full-length cDNA sequence named BTIomega1 (Buckwheat Trypsin Inhibitor) was amplified from the leaves RNA by using RT-PCR and rapid amplification of cDNA ends (RACE) methods. Sequence analysis shows that the 392 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96 and 93% homology with BWI-1 and BTI-2, a natural trypsin inhibitor from buckwheat seeds. Southern blotting suggested that three copies of BTIomega1 gene existed in the buckwheat genome. Moreover, a predicted secondary structure and 3D-structural model was constructed by homology modeling. To our knowledge, this is the first all-round report of the gene BTIomega1. The novel BTIomega1 gene has been submitted to the GeneBank under Accession No. DQ289792.     

Cloning and characterization a novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase gene AGPAT7.

The 1-Acylglycerolphosphate acyltransferase is crucial enzyme for synthesis of glycerolipids as well as triacylglylcerol biosynthesis in eukaryotes. Six members of 1-acyl-sn-glycerol-3-phosphate acyltransferase family in human have been described, which were AGPAT1, 2, 3, 4, 5 and 6. Here we report the cloning and characterization of another novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase member AGPAT7 (1-acyl-sn-glycerol-3-phosphate acyltransferase 7) gene, which was mapped to human chromosome 15q14. The AGPAT7 cDNA is 1898 bp in length, encoding a putative protein with 524 amino acid residues, which contains an acyltransferase domain in 123-234 aa. RT PCR amplification in 18 human tissues indicated that human AGPAT7 gene was widely expressed in uterus, thymus, pancreas, skeletal muscle, bladder, stomach, lung and testis. AGPAT7 protein was mainly localized to the endoplasmic reticulum (ER) in Hela cells.     

Cloning,expression and characterization of a novel human VMP gene*

We report here cloning and characterization of a novel human gene, termed VMP, which is a vesicular membrane protein. RT-PCR analysis shows that VMP is expressed exclusively in brain of the 16 tissues examined, suggesting that it is a neuron-specific membrane protein. The cDNA encodes 195 amino acid with a putative molecular weight of about 24 KDa. VMP contains two putative membrane spanning domains and a hydrophilic tail homologous to the microtubule-binding domain of MAPs. So it is speculated that VMP may associated with microtubules through its C-terminal and plays an important role in vesicular organelles transport and nerve signals.     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor.

A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

Cloning and functional characterization of a human A1 adenosine receptor.

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.