Cloning and characterization of a novel mouse short-chain dehydrogenase/reductases cDNA mHsdl2#, encoding a protein with a SDR domaid and a SCP2 domain

The short-chain dehydrogenases/reductases (SDRs) play important roles in body's metabolism. We cloned a novel mouse SDR cDNA which encodes a deduced HSD-like protein with a conserved SDR domain and a SCP2 domain. The 1.8 kb cDNA consists of 11 exons and is mapped to mouse chromosome 4B3. The corresponding gene is widely expressed in normal mouse tissues and its expression level in liver increases after inducement with cholesterol food. The predicted mouse HSDL2 protein, which has a peroxisomal target signal, is localized in the cytoplasm of NIH 3T3 cells.     

Molecular cloning and characterization of a novel human hydroxysteroid dehydrogenase-like 2 (HSDL2) cDNA from fetal brain

Hydroxysteroid dehydrogenases (HSDs) are responsible for the biosynthesis of steroid hormones and play a crucial role in mammalian physiology and development. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human hydroxysteroid dehydrogenase-like cDNA (HSDL2). This cDNA is 3211 bp in length, encoding a 418–amino-acid polypeptide, which contains a typical motif for NAD(P)⁺-binding (TGxxxGxG), an SDR active site motif (S-Y-K) and a sterol carrier protein domain. HSDL2 shows high similarity with the homologues in the mouse and fruit fly. The HSDL2 gene is mapped to chromosome 9q32 and contains 11 exons. RT-PCR analysis shows that the HSDL2 gene is widely expressed in human tissues and the expression levels in liver, kidney, prostate, testis, and ovary are relatively high.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5＇-RACE方法克隆到烟草NTHK2的全长cDNA.其全长cDNA共有3 216bp,其中5'非编码区为509bp,3＇非编码区为427bp,编码区为2 280bp,编码产物为760个氨基酸.NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域;但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代.为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域.体外激酶分析表明,当有Mg2+存在的情况下NTHK2能够自我磷酸化.进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体,参与乙烯的信号传导过程.     

Characterization of a cDNA clone encoding a basic protein, TP2, involved in chromatin condensation during spermiogenesis in the mouse

A cDNA clone encoding a small cysteine and serine-rich basic protein has been isolated from a mouse testis cDNA library. This cDNA clone encodes the mouse homologue of a protein involved in the initial phases of condensation of chromatin during spermiogenesis in rats, TP2, based on similarities in the sequence of the carboxyl terminus, composition, molecular weight, and electrophoretic mobility. Mouse TP2 can be divided into a highly basic domain comprising about one-third of the polypeptide chain at the carboxyl terminus and a much less basic domain comprising the remaining two-thirds at the amino terminus. The 5' end of the mouse TP2 mRNA contains two in-phase initiation codons both of which may be used generating two polypeptides which differ in length at the amino terminus. Southern blots demonstrate that there is a single copy of the TP2 gene in the mouse genome and Northern blots demonstrate that the polyadenylated TP2 mRNA is present at high and essentially equal levels in early and late haploid cells, and that it is virtually absent from meiotic cells.     

小鼠新分泌蛋白基因mBolA1的克隆与鉴定

采用生物信息学工具预测与实验相结合的方法得到了一个新的小鼠分泌蛋白基因mBolA1。该基因定位于染色体3F2,cDNA全长为730bp,编码137个氨基酸的蛋白,该蛋白含有一个保守的BolA结构域,等电点为9.05。用RT-PCR方法从鼠的混合cDNA库中克隆到mBolA1。Western blot实验表明mBolA1能从瞬转的COS 7细胞中分泌到细胞培养液中。亚细胞定位显示mBolA1定位于细胞浆,且与高尔基体不共定位,提示它是个非经典分泌途径的分泌蛋白。RT PCR显示mBolA1在组织中广泛表达。它的具体功能有待进一步研究。     

Promiscuous and specific phospholipid binding by domains in ZAC,a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca²⁺ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1–174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1–105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5′-ARCE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp,其中5′非编码区为509bp,3′非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域。但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代。为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域,体外激酶分析表明,当有Mg^2 存在的情况下NTHK2能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体。参与乙烯的信号传导过程。     

转录因子Cdx—2抗体的制备及对不同种属acat2启动子分析的应用

转录因子Cdx 2是一种同源盒蛋白 ,它在小肠细胞的基因表达调控和在肿瘤发生过程中起重要作用。通过融合表达小鼠Cdx 2的保守区 (mCdx 2D)、制备Cdx 2的抗体 ,进而利用该抗体检测Cdx 2在多种细胞中的表达 ,以及应用于分析不同种属Cdx 2对酰基辅酶A :胆固醇酰基转移酶 2基因 (acat2 )启动子的结合作用。首先 ,通过PCR扩增编码mCdx 2D的长 2 16bp的DNA片段 ,并将其克隆到表达质粒pGEX 4T 1中 ,构建了Cdx 2片段与GST的融合表达克隆pGEX mCdx 2D ;经SDS PAGE分析结果表明 ,融合蛋白GST mCdx 2D在大肠杆菌BL2 1(DE3)中得到可溶性表达 ;通过亲和层析分离纯化 ,获得重组表达的GST融合蛋白GST mCdx 2D。进而 ,利用该纯化的融合蛋白免疫动物制备高滴度的Cdx 2抗血清 ,经亲和吸附纯化后得到多克隆抗体。最后 ,Western印迹和EMSA实验分析结果表明 ,该抗体可用以检测不同种族 (人和鼠 )的、变性或非变性的Cdx 2 ,并显示有较好的抗Cdx 2的专一性和较高的灵敏度。同时 ,观察到Cdx 2在分化的人小肠细胞Caco 2中有高表达 ,显示具有分化依赖性 ;从抗Cdx 2抗体能识别作用于特异结合DNA的Cdx 2而形成超迁移 (supershift) ,证实了Cdx 2的确结合于不同种族 (小鼠和人 )的acat2启动子 (均存在Cdx 2元件 ) ,提示Cdx 2可能参与aca     

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.     

Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown

This report documents the characterization of a novel mouse oocyte protein which was originally identified by microsequence analysis of a 67.8 kDa protein spot (pI 5.7) on a Coomassie-stained two-dimensional (2D) gel of murine egg proteins. Tandem mass spectroscopic analysis of the peptides obtained from the cored protein yielded sequences that appeared to match only ovary, egg, and preimplantation embryo cDNAs. We then cloned the novel gene by RACE-PCR, and analysis of the deduced cDNA sequence found that this maternal product was ∼56% identical to human cytosolic phospholipase A2γ (cPLA2γ). Based on this sequence homology, we named the molecule mouse cytosolic phospholipase A2γ (cPLA2γ). As with human cPLA2γ, mouse cPLA2γ contains a lipase consensus sequence and lacks the calcium binding domain that is found in other PLA2 proteins. However, mouse cPLA2γ is different from human cPLA2γ in that mouse cPLA2γ expression is restricted to the ovary and that the protein does not contain the myristoylation and prenylation lipid-anchoring motifs that are present in human cPLA2γ. Within oocytes, mouse cPLA2γ localizes mainly to the oocyte cortex and to the nucleoplasm. Interestingly, during germinal vesicle breakdown, mouse cPLA2γ aggregates dynamically relocate from the oocyte cortex to the nuclear envelope, suggesting a possible role for this putative egg-restricted phospholipase A2γ in membrane remodeling. Furthermore, mouse cPLA2γ protein continues to be expressed in the embryo until the 4-8-cell stage of development, suggesting that mouse cPLA2γ may function as a previously uncharacterized maternal effect gene.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.