Molecular cloning and expression of a new gene,GON-SJTU1 in the rat testis

Background

A prolonged latent phase is independently associated with an increased incidence of subsequent labor abnormalities. We aimed to compare between oxytocin augmentation, amniotomy and a combination of both on the duration of labor among women with a prolonged latent phase.

Methods

Women with a singleton fetus in cephalic presentation who have a prolonged latent phase, were randomly allocated to amniotomy (group 1), oxytocin (group 2) or both (group 3). A group of women who progressed spontaneously without intervention composed the control group (group 4). The primary outcome was the duration of time from initiation of augmentation until delivery.

Results

A total of 213 women were consented and randomized to group 1 (70 women), group 2 (72 women) and group 3 (71 women). Group 4 was composed from additional 70 women. A mean reduction of 120 minutes in labor duration was observed among group 3 compared to group 1 (p = 0.08) and 180 minutes compared to group 2 and 4 (p = 0.001). Women in group 3 had a shorter length of time from augmentation until the beginning of the active phase and a shorter first stage of labor than group 1 (p = 0.03), group 2 (p = 0.001) and group 4 (p = 0.001). Satisfaction was greater among group 3 and 4. Mode of delivery and neonatal outcome were comparable between the groups.

Conclusion

Labor augmentation by combined amniotomy and oxytocin among women with a prolonged latent phase at term seems superior compared to either of them alone.     

Molecular cloning and characterization of a new gene, Oocyte-G1

Oocytes are recognized as a source of regulatory molecules that influence follicular development through an array of actions on granulosa cells. Recently, more and more hormones and signaling molecules were identified during follicular developmental processes; however, the details about their functions are still unclear. During efforts to clone follicular development-related genes, we isolated a cDNA fragment by DDRT-PCR. To obtain cDNA 5'- and 3'-end sequences, we screened a mouse ovarian cDNA library. After screening the library, an open reading frame of 2,994 bp for the new gene (Oocyte-G1), which encodes a 997-residue protein, was cloned. Northern blot analysis revealed the presence of approximately 3.6 kb Oocyte-G1 mRNA in ovary, lung, kidney, testis and brain. Northern analysis of RNA from ovaries in vivo showed that Oocyte-G1 was weakly expressed on day 5 and at a moderate level on day 10. Thereafter, on day 15 or in adults (day 40), there was an increase in expression, followed by a decline in ovaries on day 20 or older (day 120). Furthermore, we studied the Ooctye-G1 protein by using the antiserum against a peptide sequence unique to this gene in Western blotting and immunolocalization. The antiserum recognized a prominent band of approximately 110 kDa in immunoblots and signals were dispersed in oocytes and some cumulus granulosa cells. Our results suggest the potential role of Oocyte-G1 in ovarian follicular development.     

Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

[目的]从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质.[方法]通过多序列比对设计简并引物,扩增出真菌V. dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列.经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63 bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA.将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析.[结果]BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%.测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果.Mg2 和Ca2 对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%.[结论]本文从引起棉花黄萎病的真菌V. dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列.本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作.酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值.     

Molecular cloning and developmental expression of AtGR1, a new growth-related Arabidopsis gene strongly induced by ionizing radiation

Screening for mRNAs that accumulate after DNA damage induced by ionizing radiation, we have isolated a 2.0-kb cDNA coding for a new Arabidopsis PEST-box protein named AtGR1 (A. thaliana gamma response 1) with an expression profile similar to that observed for several plant cell cycle-related proteins. Using an anti-AtGR1 antibody, we have shown that the AtGR1 protein is expressed at basal levels in mitotically dividing cells (meristematic tissues and organ primordia) and at a strongly enhanced level in endoreduplicating cells (stipules, trichomes). Using transgenic Arabidopsis plants that express the GUS reporter gene under the control of the AtGR1 promoter, we have demonstrated that the observed AtGR1 protein distribution is due to the promoter activity. Our results suggest that basal AtGR1 levels are associated with progression through mitosis, whereas elevated intracellular levels of AtGR1 seem to induce changes between the S and M phases of the cell cycle that trigger somatic cells to enter the endoreduplication cycle. Ionizing radiation-induced rapid and dose-dependent accumulation of AtGR1 mRNA in cell cultures and plant tissues leads to tissue-specific accumulation of AtGR1 protein, best observed in ovules, which never undergo an endoreduplication cycle. It therefore appears that the radiation-induced transient AtGR1 accumulation reflects DNA damage-dependent transient cell cycle arrest before mitosis, which is necessary to accomplish DNA repair prior to chromosome segregation and cytokinesis.     

Molecular cloning, expression and characterization of the zebrafish bram1 gene, a BMP receptor-associated molecule

Summary We have identified a cDNA clone encoding BMP receptor-associated molecule 1 (BRAM1) from the zebrafish expressed sequence tag (EST) database. The 2606 bp full-length bram1 cDNA was cloned, and further confirmed by nucleotide sequencing. The zebrafish sequence encodes a protein of 195 amino acids with an evolutionarily conserved MYND domain, which displays ∼ ∼98% homology with human and mouse BRAM1, and ∼ ∼64% homology with C. elegans BRA-1 and BRA-2. The bram1 gene, composed of five exons and four introns, spans ∼ ∼14 kb on linkage group 14 of the zebrafish genome. RT-PCR and whole mount in situ hybridization analyses disclosed that zebrafish BRAM1 is a maternal factor. The protein interacts directly with zebrafish BMP Receptor type IA, as observed from GST-pull down and co-immunoprecipitation assays. Furthermore, cotransfection of zebrafish BRAM1 with the corresponding BMP receptor resulted in down-regulation of BMP-mediated signaling. Our results collectively indicate that BRAM1 plays a biological role during zebrafish development.     

绵羊CCNG1基因克隆及表达分析

细胞周期蛋白cyclin G1(CCNG1)是一个重要的细胞周期调控因子,参与哺乳动物颗粒细胞增殖、卵母细胞成熟等繁殖生物学过程,但其在绵羊中鲜有报道。为了研究CCNG1基因对绵羊发情调控以及季节性繁殖的影响,文章对绵羊CCNG1基因进行了克隆和组织表达谱分析,利用Real-time PCR对该基因在多浪羊(常年发情)与中国美利奴羊(季节性繁殖)发情周期不同阶段性腺轴组织的表达变化进行了实时检测。获得了绵羊CCNG1基因部分cDNA序列,其中编码区全长885 bp,编码294个氨基酸。CCNG1蛋白结构经预测存在多个磷酸化位点和蛋白激酶C磷酸化位点。CCNG1基因在所检测绵羊各组织中均有表达,但在卵巢与肾脏中为高丰度表达;CCNG1在不同绵羊品种发情周期不同阶段性腺轴组织的表达变化规律基本一致,卵巢、子宫、松果体、垂体均是在发情期达到峰值。但是在发情期和发情后期卵巢CCNG1的表达量存在显著的品种间差异(P<0.01)。研究结果表明,CCNG1可能通过参与卵泡的生长发育继而达到对绵羊发情和季节性繁殖的调控。     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

Rat ceruloplasmin. Molecular cloning and gene expression in liver, choroid plexus, yolk sac, placenta, and testis

A rat ceruloplasmin cDNA clone was isolated from a rat liver cDNA library and identified by partial nucleotide sequence analysis. Rat liver ceruloplasmin mRNA levels were measured during the acute phase response to inflammation by cytoplasmic dot hybridization to ceruloplasmin cDNA. Regulation of ceruloplasmin synthesis appeared to be at the mRNA level, with the concentration of ceruloplasmin mRNA increasing significantly 12 h after induction of inflammation, reaching a maximum of 350% of normal at 36 h and returning to normal levels within 60 h. Using Northern blot analysis, extrahepatic ceruloplasmin gene expression was observed in choroid plexus, yolk sac, placenta, and testis. All these tissues are at the interface between, and possibly involved in maintaining homeostasis in, adjacent extracellular compartments. No ceruloplasmin mRNA was detected in RNA from stomach and small intestine.     

Molecular cloning and expression analysis of a new stress-related AREB gene from Arachis hypogaea

An AREB gene, designated as AhAREB1, was cloned from peanut (Arachis hypogaea L.). The gene contains a 1 338-bp open reading frame that encodes a putative protein of 445 amino acids. The corresponding genomic DNA containing four exons and three introns was isolated and analyzed. An upstream 1 060-bp DNA promoter fragment of the AhAREB1 gene was also amplified from peanut genomic DNA. Multiple sequence alignment of the deduced amino acids of AREB showed that the AhAREB1 protein shares high sequence homology with GmAREB1, S1AREB and ABF2. Quantitative real-time PCR analysis showed that AhAREB1 was induced by polyethylene glycol, NaCl, gibberellic acid, abscisic acid and salicylic acid. The cloning and characterization of the AhAREB1 gene will be useful for further studies establishing the biological role of AhAREB1 in plants.     

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka,Oryzias latipes

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.     

大鼠OB基因克隆及其在大肠杆菌中的表达

采用ＲＴＰＣＲ技术扩增大鼠ＯＢｃＤＮＡ编码区序列。ＰＣＲ产物酶切后定向克隆至ｐＵＣ１９质粒。经核苷酸序列测定表明与文献报道的大鼠ＯＢｃＤＮＡ编码区序列一致。继之构建了ｐＢＶ２２０ｒＯＢ表达质粒并获得了ＯＢ基因在大肠杆菌中的特异表达。大鼠ＯＢ基因产物的获得为研究肥胖与某些非传染性疾病（如糖尿病、高血压病）间的关系提供了条件     

Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3′-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals. Received: 5 May 1999 / Accepted: 11 February 2000     

Molecular cloning of a new endoglucanase gene from Clostridium cellulolyticum and its expression in Escherichia coli

Summary Two genes coding for endoglucanase activity in Clostridium cellulolyticum were cloned and expressed in Escherichia coli by using plasmid pUC18. The sizes of two fragments harbouring endoglucanase genes are 4.4 kb and 2.0 kb, respectively. The 2.0-kb fragment was identical with a reported DNA fragment encoding an endoglucanase of C. cellulolyticum. The 4.4-kb fragment was obtained first in this study. Deletion analysis showed that a 1.3-kb portion of the 4.4-kb fragment is necessary for the endoglucanase expression by its own promoter. The 4.4-kb fragment hybridized with several different fragments of the genomic DNA in C. cellulolyticum.Offprint requests to: T. Kodama