Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf

The sequence of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mRNA from maize has been determined. Complementary DNAs were isolated from a cDNA library of light-induced leaf poly(A)+ RNA constructed in an expression vector. An open reading frame beginning at an ATG codon at nucleotide 328 of the longest cDNA (5617-bases long) encoded 1616 amino acid residues. The amino terminus of the purified mature enzyme coincided with the cysteine residue at position 98 of the predicted sequence. This enzyme is homologous with the large subunit of Escherichia coli NADPH-dependent glutamate synthase having about 42% identical residues between the two proteins. The enzyme also contains a short region similar to a potential FMN-binding region of yeast flavocytochrome b2. The cDNA hybridizes to an RNA band about 5.5 kilobases whose steady-state level is markedly increased upon illumination of etiolated maize seedlings. Analysis of genomic DNA indicates the presence of a single-copy gene for ferredoxin glutamate synthase in maize.     

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Molecular cloning of DNA sequences coding for mouse embryonic globins

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not share any of the studied restriction sites with the other plasmids, while no. 2 and 54 have at least one site in common. The coding properties of inserted DNA were determined by positive hybrid translation showing that no. 2 codes for the alpha-like embryonic chain x, while no. 16 and 54 code for a beta-like embryonic chain, either y or z.     

Molecular cloning of the complementary DNA for a human folate binding protein

The complementary DNA for a human folate binding protein has been cloned from a lambda gt11-cDNA library prepared from cultured KB cells. A number of clones were selected by immunoscreening with a monospecific antiserum and by oligonucleotide probes corresponding to the NH2-terminal sequence of the folate binding protein. A partial nucleotide sequence of the cDNA was determined directly from the lambda gt11 phage and after subcloning into M13. The 18 amino acids deduced from the initial 19 codons were exactly the same as the amino acid sequence obtained by peptide analysis of the purified protein providing proof that this clone is the folate binding protein cDNA.     

Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA

Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.     

Molecular cloning and characterization of the murine bile salt export pump

Hepatic bile salt secretion and bile formation are essential functions of the mammalian liver, and the rate-limiting step of hepatocellular secretion of bile salts is canalicular secretion. Recently, the rat sister-of-p-glycoprotein/bile salt export pump (spgp/BSEP) was demonstrated to encode for the rat ATP-dependent canalicular bile salt export protein, and mutations of human BSEP were identified as the cause of PFIC 2. Since mouse models are vital for studies in hepatocellular transport and metabolism, cloning and characterization of the murine gene are essential. In this study, we have cloned a full-length, functional cDNA for the mBsep. The deduced amino acid sequence encodes for a 1321-amino-acid protein and is 94% similar to rat and 89% similar to human bsep. Western immunoblotting using an antibody directed against a carboxy-terminal peptide of mbsep protein reveals a 160kDa protein, which is highly enriched in mouse canalicular membranes. Transfection of mBSEP into Sf-9 insect cells or mammalian Balb-3T3 cells confers functional transport of the bile salt taurocholate. The mBsep mRNA is expressed in murine liver, but not in other tissues. Hepatic mBsep levels appear highly regulated, being markedly diminished in both LPS and estrogen models of cholestasis. These data are important for further murine studies of hepatocellular transport physiology and metabolism.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.     

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.     

Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1

Mutations in the GDD1 gene cause gnathodiaphyseal dysplasia, a rare human skeletal syndrome with autosomal dominant inheritance. The biochemical function(s) of GDD1 protein and the molecular pathophysiology of GDD1 mutations leading to GDD have not yet been elucidated. In this study, we characterized the complete cDNA sequence and genomic organization of the mouse GDD1 gene. Analysis of GDD1 mRNA revealed a complex alternative splicing pattern, involving five exons of the GDD1 gene. GDD1 isoforms lacking conserved amino acids at the N-terminus cytoplasmic tails, and with changes in transmembrane topology, are presumably associated with changes in protein functions and subcellular localizations of GDD1. We found GDD1 expression to be up-regulated during the course of myogenic differentiation in the murine pluripotent mesenchymal precursor cell line C2C12, whereas its expression was diminished during osteoblastic differentiation. These observations suggest diverse cellular roles of GDD1 protein.     

Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.