GEC1, a protein related to GABARAP, interacts with tubulin and GABA(A) receptor

We have previously identified in uterine cells a novel estrogen-regulated gene called gec1. GEC1 presents 87% identity with GABARAP which, so far, was the only protein found to associate with tubulin and GABA(A) receptor. We demonstrated then that GEC1 interacts in vitro with tubulin and GABA(A) receptor, and promotes tubulin assembly and microtubule bundling. Since all polyclonal antibodies failed in discrimination of both proteins GEC1 and GABARAP, a GEC1-GFP fusion protein was used to specifically localize GEC1. GEC1-GFP was distributed over the cytoplasm in perinuclear vesicles with a scattered pattern. Overall, our data show that GEC1 could be a new member of the GABARAP family involved in the transport of GABA(A) receptor.     

家兔BMP7基因的克隆及其生物信息学分析

在对已知部分编码序列(CDS)进行分析的基础上, 采用RT-PCR分步扩增以及RACE方法, 对家兔BMP7基因3′和5′末端未知序列进行了克隆与生物信息学分析。测序结果综合分析表明, 所获序列共计1 654 bp, 包括家兔BMP7近全长前肽、全长成熟肽CDS及3′非翻译序列(3′UTR), 将已有的序列向5′和3′端分别延伸了395 bp和628 bp。序列对比表明, 克隆的家兔BMP7 CDS部分与人、小鼠的对应序列的同源性分别为91.89%和89.32%, 预测的氨基酸序列同源性分别为96.51%和96.01%。家兔BMP7 3′UTR长446 bp, 与人、小鼠对应序列同源性分别为57.38%和45.57%; 具有2个转录终止信号位点。推测家兔BMP7成熟蛋白有BMPs特有的7个位置固定的半胱氨酸残基和TGF-β家族指纹。家兔BMP7 3′UTR区转录终止信号的可选择性可能与基因转录后调控有关。     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.

A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.     

Molecular cloning of LMO41, a new human LIM domain gene

We have identified a new human LIM domain gene by isolating an autoantigenic cDNA clone from a human breast tumor cDNA library. The predicted amino acid sequence of the cDNA clone's 495 bp open reading frame contains two tandem LIM domain motifs, and within the LIM domain region there is 62% identity with the analogous region of the LIM-only gene LMO1. The homology to LMO1 is restricted to the 360 bp region encoding the tandemly repeated LIM domains, the rest of the open reading frame as well as the extensive, GC-rich 5' untranslated region, and 3' region of the 2 kb cDNA sequence are unrelated to any known genes. This gene has been designated LMO4.     

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.     

人SBK1 cDNA的克隆及其相互作用蛋白的筛选

首次克隆到人的SBK1（homo sapiens SH3-binding domain kinase 1,SBK1）的cDNA序列,并通过生物信息学的手段,电子克隆到人SBK1的基因组DNA序列.人的SBK1是鼠SBK1的直系同源物,两者基因组DNA结构相似,均含有4个外显子.人的sbk1基因ORF长1 275 bp,编码424个氨基酸,而鼠的ORF长1 254 bp,编码417个氨基酸.两者编码区的核苷酸序列同源性达87.7%,而氨基酸序列同源性达95.7%,在羧基端均有一个PV富集区,推测其能与含有SH3结构域的蛋白质结合.将RT－PCR所获得的长度为1 610 bp的sbk1cDNA序列搜索EST数据库,进行电子延伸,最终获得了约5 kb的人sbk1全长mRNA序列,它与鼠的sbk1全长mRNA大小一致;通过比较基因组学发现UniGene族Hs.97837实际上代表了sbk1基因UniGene族Hs.460471的3′UTR区域,而不是代表了一个新的UniGene族.采用酵母双杂交技术,以SBK1为“诱饵”,获得了与之相互结合的蛋白表皮生长因子受体EGFR和核孤儿受体蛋白NR4A1,它们之间的具体功能关系有待进一步研究.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.