Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.     

Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs

Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.     

The molecular cloning and expression of two CRABP cDNAs from human skin

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.     

Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung.

Complementary DNA clones corresponding to the 70- and 82-kDa subunits of soluble guanylate cyclase of rat lung have been isolated. Blot hybridization of total poly(A)+ RNA from rat tissues detected mRNA of about 3.4 kilobases for the 70-kDa subunit and about 5.5 kilobases for the 82-kDa subunit. Messenger RNA levels of both subunits were abundant in lung and cerebrum, moderate in cerebellum, heart, and kidney, and low in liver and muscle, consistent with previously described enzyme activities in these tissues. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated that the genes for the 70- and 82-kDa subunits are different. The carboxyl-terminal region of the 70- and 82-kDa subunits showed a high degree of homology and also had a partial homology with the putative catalytic domain of particulate guanylate cyclase and adenylate cyclase, indicating that both the 70- and 82-kDa subunits have catalytic domains. The cDNAs were subcloned to an expression vector and transfected to L cells. The cells transfected with cDNA of the 70-kDa subunit or the 82-kDa subunit showed no guanylate cyclase activity, whereas the cells transfected with both the 70- and 82-kDa subunit cDNAs showed significant guanylate cyclase activity that was activated markedly by sodium nitroprusside. These data suggest that both subunits are required for both the basal catalytic and regulatory activity of soluble guanylate cyclase. Presumably both catalytic subunits must be present and interactive to permit synthesis of cyclic GMP and nitrovasodilator activation.     

Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.     

Molecular cloning of two species of cDNAs for human alpha-N-acetylgalactosaminidase and expression in mammalian cells

Two species of cDNAs for human alpha-N-acetylgalactosaminidase were isolated from a human fibroblast cDNA library. The two species differ each other by a 70 bp insertion in the coding region. Transient expression study in COS cells demonstrated that only the cDNA without the 70 bp insertion expressed alpha-N-acetylgalactosaminidase activity. Analysis of mRNA species utilizing polymerase chain reaction revealed that the majority of the mRNA does not contain the 70 bp insertion, and the mRNA containing the 70 bp insertion is present only in a minor amount in human brain.     

Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells

We report the isolation and characterization of two human liver cDNA clones, HUG-Br1 and HUG-Br2; each encodes a UDP-glucuronosyltransferase enzyme which glucuronidates bilirubin IX alpha to form both the IX alpha C8 and IX alpha C12 monoconjugates and a diconjugate. HUG-Br1 cDNA (2351 base pairs) and HUG-Br2 cDNA (2368 base pairs) encode proteins with 533 and 534 amino acid residues, respectively, with a typical membrane-insertion signal peptide, membrane-spanning domain, and 3 or 5 potential asparagine-linked glycosylation sites. At the nucleic acid and deduced amino acid sequence levels the two clones are 82% similar overall, 66% similar in the amino termini, and identical after codon 287, thus encoding proteins with the same carboxyl terminus. The mRNA encoding HUG-Br1 is of high abundance, and the one encoding HUG-Br2 is of low abundance; both are 2.6 kilobases in length. Both messages (2.6 kilobases) were present in the explanted liver of a Type I Crigler-Najjar patient, although the level for that of HUG-Br1 was reduced 4.5-fold. Northern blot analysis of poly(A)+ RNA isolated from the liver of an untreated and a phenobarbital-treated Erythrocebus patas monkey with 5'-specific probes for each clone indicated that the HUG-Br2-encoded message is induced two fold, but that for HUG-Br1 is not. These data indicate that bilirubin is glucuronidated by at least two different proteins, most likely present in very different amounts. These cDNAs which encode functional bilirubin UDP-glucuronosyltransferases will allow the isolation of an appropriate gene to develop a gene therapy model for patients which have the totally deficient trait.     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.     

Cloning and expression of multiple protein kinase C cDNAs

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.     

凝血系统相关基因突变及表达异常与高血凝

摘要高血凝是动脉粥样硬化(As)的危险因子,在As的发展中具有重要作用。凝血系统、抗凝系统、纤溶系统及其它相关基因的突变及表达异常导致高血凝的产生。凝血系统的凝血因子V基因、凝血酶原基因、组织因子基因,抗凝系统的血栓调节蛋白基因、抗凝血酶Ⅲ基因,纤溶系统的纤溶酶原激活物抑制剂-1基因,均与高血凝密切相关。     

Cross induced coagulations between human and crustacean clotting factors. Considerations on the clotting processes

1. Cross induced coagulations show that human factor XIII and crustacean coagulin are to some extent functionally equivalent and may be substituted for each other. 2. In crustacea, fibrinogen and coagulin appear as in situ activated products since they are both able to react with non-activated human clotting factors. 3. The coagulin catalyzed transamidation which stabilizes the clot and renders it insoluble in 1-5% monochloroacetic acid solutions seems to be the basic reaction of the clotting process in the animals in which coagulation occurs. 4. The possibility of a two step clotting in crustacea is discussed.