Cloning and characterization of mouse Dhm2 cDNA,a functional homolog of budding yeast SEP1

We have isolated mouse Dhm2 cDNAs encoding a homolog of budding yeast SEP1, whose product is involved in many cellular processes including meiosis, cellular senescence, and telomere maintenance. The putative Dhm2 protein (Dhm2p), which consists of 1687 amino acids and whose molecular weight is 191 400, matches the size of Sep1p and shares extensive homology with Sep1p especially in their N-terminal regions. A multicopy plasmid containing of the Dhm2 cDNA complements the slow growth phenotype, sporulation defect, and DNA recombination defect caused by the sep1 mutation in yeast, indicating that Dhm2 is a functional homolog of SEP1. Since Dhm1, another SEP1 homolog we reported previously, only partially compensates for the sep1 mutation, we conclude that Dhm2 is a true homolog of SEP1. Northern analysis revealed that 5.8 kb mRNA corresponding to Dhm2 open reading frame is produced highly in testis. These results strongly suggest that Dhm2p participates in gametogenesis in mouse.     

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 identification of a novel human gene PDLIM5, a homolog of AD-associated neuronal thread protein (AD7c-NTP).

A novel human gene cDNA was successfully cloned from the human fetal brain cDNA library constructed by our lab, and this gene was termed PDLIM5 after acquiring the agreement of HUGO. BLASTX searching revealed that the hypothetical protein is a homolog of AD-associated neuronal thread protein (AD7c-NTP), which is over-expressed in Alzheimer disease (AD) beginning early in the course of disease, and over-expression of the AD7c-NTP gene would cause neuritic sprouting and cell death. SMART analysis showed that both our predicted protein and AD7c-NTP comprise BCL domain (only contains BH1 and BH2 regions). RT-PCR experiment revealed that the expression level of PDLIM5 in brain, skeletal muscle, prostate, colon and leukocyte is obviously higher than that in other tissues.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.

We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.     

Cloning of the gene and cDNA for human heart chymase

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.     

Cloning of the cDNA for a human homolog of the rat PEP-19 gene and mapping to chromosome 21q22.2–q22.3

Exon trapping was used to identify fragments of genes on human chromosome 21. One trapped sequence, hmc18h10 (GenBank no. X88329), showed homology to a sequence (GenBank no. S65225) that includes the first three codons of the rat PEP-19 gene and 5′ untranslated leader region. We have cloned the corresponding cDNA for a human homolog of the rat PEP-19 gene and mapped it to the region between markers ERG and D21S56 of chromosome 21q22.2–q22.3. Rat PEP-19 is a neuron-specific polypeptide expressed in several regions of the central nervous system. It serves as a cell-specific marker in Purkinje cells and its expression is developmentally regulated in the cerebellum, but its precise function is unknown. It is also presently unknown whether overexpression of the PEP-19 gene is involved in certain phenotypes of Down syndrome. Received: 3 May 1996 / Revised: 2 July 1996     

Cloning the polB gene of Escherichia coli and identification of its product

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.     

Cloning and identification of a novel cDNA coding thioredoxin-related transmembrane protein 2

Thioredoxin plays an important role in various cellular processes through redox regulation. Here we report the molecular cloning and characterization of one member of the thioredoxin superfamily, designated as TMX2. The TMX2 cDNA consists of 1644 nucleotides and contains an open reading frame encoding a protein of 372 amino acids with a predicted molecular mass of 42.5 kDa and an isoelectric point of 8.94 . The TMX2 protein may possess an N-terminal signal peptide, a potential transmembrane domain, an Myb DNA-binding domain repeat signature, a thioredoxin consensus pattern, an endoplasmic reticulum (ER) membrane retention signal (KKXX-like motif), and a dileucine motif in the tail. Northern blot analysis shows it is widely expressed in human tissues.     

Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant.

Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis. A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae. The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb. The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively. In contrast, it has only 21% identity with the S. cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism.