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PubMed1989

Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7.

The cDNA containing the full coding sequence of human NADPH-P450 oxidoreductase was isolated and completely sequenced. The cDNA contained 2398 base pairs, including 9 and 358 base pairs of 5' and 3' noncoding sequences, respectively. The human NADPH-P450 oxidoreductase protein deduced from the cDNA has 677 amino acids, with a calculated molecular weight of 76,656. The cDNA nucleotide and deduced amino acid sequences displayed 83 and 92% similarities, respectively, with those of the rat NADPH-P450 oxidoreductase. By use of somatic cell hybrids, the NADPH-P450 oxidoreductase gene was regionally localized to human chromosome 7 (7p15-q35). The levels of NADPH-P450 oxidoreductase protein and mRNA were analyzed in 13 human liver specimens and less than 3-fold variation was found among the different livers. The NADPH-P450 oxidoreductase cDNA was inserted into vaccinia virus and expressed in cell culture. The cDNA-expressed enzyme was active in reducing the electron acceptor cytochrome c. In addition, the NADPH-P450 oxidoreductase stimulated the enzymatic activity of vaccinia virus-expressed human P3(450) when both recombinant viruses were used to coinfect human cells in culture. An approximate equal mole level of NADPH-P450 oxidoreductase and P3(450) was required to achieve maximal activity for both ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase.

Animals↗
PubMed1989

Complementary DNA encoding core protein II of human mitochondrial cytochrome bc1 complex. Substantial diversity in deduced primary structure from its yeast counterpart.

Core protein II of mitochondrial cytochrome bc1 complex is the second largest nuclear-encoded subunit that is essential for the assembly of the functional complex. We have isolated, for the first time, a cDNA clone for a mammalian core protein II. Immunoscreening of a lambda gt11 rat liver cDNA library resulted in isolation of a cDNA encoding a partial sequence of rat mitochondrial core protein II, and this cloned cDNA was used as a probe for colony hybridization to screen a human fibroblast cDNA library. By comparison with the known NH2-terminal amino acid sequence of bovine core protein II, the amino acid sequence deduced from a cloned human cDNA was predicted to be composed of the part for the NH2-terminal extension of 14 amino acid residues and that for the mature polypeptide of 439 residues. The presequence is typical of precursors to mitochondrial proteins in being composed of a combination neutral and basic residues. The deduced sequence shows rather low homology with that of the corresponding subunit of yeast cytochrome bc1 complex; this finding is in contrast with the relatively high homology observed for the sequences of the redox center-carrying subunits of the same complex. On Northern blot analysis of human fibroblast poly(A)+ RNA, the cDNA hybridized to two mRNAs, a major species of 1700 nucleotides and a minor one of 2000 nucleotides. Southern blot hybridization of genomic DNA suggested that the human genome contains one or two genes for core protein II.

Amino Acid Sequence↗
PubMed1989

Identification of major autolytic cleavage sites in the regulatory subunit of vascular calpain II. A comparison of partial amino-terminal sequences to deduced sequence from complementary DNA.

Purified calpain II from vascular smooth muscle is a heterodimer consisting of catalytic (Mr = 76,000) and regulatory (Mr = 30,000) subunits. In the presence of Ca2+, the regulatory subunit undergoes stepwise autolysis resulting in enzyme activation. By slowing autoproteolysis, we identified major autolytic intermediates of the regulatory subunit. Gas-phase sequencing of the regulatory subunit and its autolytic fragments revealed that the NH2-terminus of the Mr = 30,000 form was blocked, whereas each fragment yielded a unique amino acid sequence, suggesting that autolysis proceeds in an NH2- to COOH-terminal direction. By comparison of actual amino acid sequences of autolytic cleavage intermediates to the full sequence deduced from cDNA, we have identified the major autolytic cleavage sites. Three different peptide bonds were cleaved, with neutral amino acids predominating on both sides of the peptide bond hydrolyzed. Importantly, leucine or isoleucine was identified in the second position upstream from the cleavage site in all three autolytic sequences. The presence of an upstream leucine residue in the autolytic cleavage sequence is reminiscent of the structure of potent microbial and synthetic peptide inhibitors of calpain.

Amino Acid Sequence↗
PubMed1989

[Prolactin receptor: characterization by monoclonal antibodies and cloning of complementary DNA].

Rat liver prolactin receptor has been partially characterized and purified to homogeneity using monoclonal antibodies. Pure receptor was digested with trypsin and amino acid sequence of receptor fragments determined. This allowed us to clone the prolactin receptor cDNA. Our approach to clone the receptor cDNA consisted of synthesizing oligonucleotides corresponding to the amino acid sequence of receptor fragments, and to screen a cDNA library. Sequencing reveals that prolactin receptor is a 291 amino acid protein, containing an extracellular domain of 210 residues, a single transmembrane segment of 24 amino acids and a cytoplasmic domain of 57 amino acids. Introduction of the prolactin receptor cDNA into various cell types demonstrates that the single protein is sufficient to bind prolactin with the same affinity and specificity reported for the prolactin receptor.

Animals↗
PubMed1988

Molecular cloning of complementary DNA to mouse tissue plasminogen activator mRNA and its expression during F9 teratocarcinoma cell differentiation.

Two tissue plasminogen activator (tPA) cDNA clones were isolated from mouse cDNA libraries and characterized. The sequence coding for mature mouse tPA protein including its 29-residue signal peptide, as well as that for the 3' and part of the 5'-nontranslated regions of the mRNA was determined. The mature protein consists of 530 amino acids and is encoded by a transcript of approximately 2800 nucleotides. The mouse tPA protein has 81% homology with its human counterpart, and amino acid sequence conservations suggest that the multidomain-like structure found for human tPA is maintained in the mouse enzyme. The tPA cDNA has been used as a probe to analyze tPA gene expression during F9 cell differentiation. tPA mRNA is not detected in F9 stem cells. When F9 cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP, tPA mRNA accumulates. We demonstrate that this induction occurs at least in part because of an increase in tPA gene transcription.

Animals↗
PubMed1988

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex.

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase).

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1986

Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage.

A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma.

Cloning, Molecular↗
PubMed1986

Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone.

We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.

Alleles↗
PubMed1987

Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family.

Lauric acid omega-hydroxylase (cytochrome P-450LA omega) was purified from livers of rats that had been given the hypolipidemic drug clofibrate. Immunoblot analysis with anti-cytochrome P-450LA omega antibody revealed the presence of two clofibrate-induced cytochrome P-450 proteins in both liver and kidney. This antibody was used to screen a lambda gt11 expression library constructed from liver mRNA isolated from rats given clofibrate. The clone, pP-450LA omega, was isolated and found to contain 2062 base pairs with an open reading frame of 509 amino acids (Mr 58,222). The pP-450LA omega cDNA insert was placed in the yeast expression vector pAAH5, and the resultant plasmid expressed a protein of the same size and activity as cytochrome P-450LA omega. The mechanism of the regulation of the cytochrome P-450LA omega gene by hypolipidemic agents was examined. The rate of cytochrome P-450LA omega gene transcription was increased as early as 1 h after administration of clofibrate, and this increase was followed by an elevation of cytochrome P-450LA omega mRNA, immunochemically detectable protein, and cytochrome P-450LA omega activity. In contrast, no increase in transcriptional activity was detected after clofibrate administration for the cytochrome P-450PCN or P-450b/e genes. The cytochrome P-450LA omega has several structural features in common with other cytochrome P-450s, including a conserved cysteine-containing heme-binding fifth ligand fragment and a hydrophobic amino terminus. Overall, cytochrome P-450LA omega shared less than 35% cDNA nucleotide and amino acid similarity with cytochromes P-450c, P-450d, P-450e, and P-450PCN, indicating that it is a member of a new cytochrome P-450 gene family. Southern blot analysis indicates that this family contains two or three genes.

Amino Acid Sequence↗
PubMed1987

The G protein beta 2 complementary DNA encodes the beta 35 subunit.

Antisera were generated against synthetic peptides that correspond to amino acid sequences deduced from a cDNA (designated beta 2) that encodes a second form of the beta subunit of guanine nucleotide-binding regulatory proteins (G proteins). The specificity of interactions of these antisera with purified G protein beta subunits indicates that the beta 2 cDNA encodes the beta 35 form of this polypeptide. This hypothesis is confirmed by the use of these antisera to detect expression of the beta 2 cDNA in COS-m6 cells.

Base Sequence↗
PubMed1988

Identification of the GTP-binding protein encoded by Gi3 complementary DNA.

Three closely related, but distinct, GTP-binding proteins (G-proteins) are encoded by cDNAs arbitrarily designated Gi1, Gi2, and Gi3. The in vitro translated products of mRNAs prepared from Gi1, Gi2, and Gi3 cDNAs migrate as 41-, 40-, and 41-kDa proteins, respectively, on sodium dodecyl sulfate-polyacrylamide gels. Antisera were raised against synthetic decapeptides corresponding to a divergent sequence (residues 159-168 for Gi1 and Gi3; 160-169 for Gi2) of the three cDNAs and tested on immunoblots for reactivity with three purified G-proteins, G41 and G40 from brain and G41 from HL-60 cells. LD antisera (Gi1 peptide) react only with brain G41. LE antisera (Gi2 peptide) react only with brain G40, and SQ antisera (Gi3 peptide) react exclusively with HL-60 G41. The results indicate that the 41-kDa G-protein purified from HL-60 cells differs from the purified brain 41-kDa protein and suggest that the HL-60 cell protein corresponds to that encoded by Gi3 cDNA.

Amino Acid Sequence↗
PubMed1977

Synthesis of RNA from cellulose-bound complementary DNA.

Radioactively labeled RNAs were synthesized from cellulose-bound cDNA templates using Escherichia coli RNA polymerase. Hybridization of this RNA to excess unlabeled cDNA approached 100%, indicating the complementarity of product and template. The average length of the RNA product, as determined by formamide gels, was approximately 40% of the template length. Hybridization of unlabeled globin RNA produced by this technique to labeled globin cDNA indicated the population of RNA sequences represented at least 80% of the template sequences. Approximately 30% of the RNA product by mass contains poly(A) tails as determined by binding to oligo(dT)-cellulose. The template can be reused for several cycles of synthesis with little loss of synthetic capability and therefore, can amplify the amount of mRNA initially used to produce the template.

Cellulose↗
PubMed1988

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis.

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.

Amino Acid Sequence↗
PubMed1988

Carbohydrate binding protein 35. Complementary DNA sequence reveals homology with proteins of the heterogeneous nuclear RNP.

The complete nucleotide sequence of a cDNA clone for carbohydrate binding protein 35, a galactose-specific lectin identified in the nucleus of mouse 3T3 fibroblasts, has been determined. The deduced amino acid sequence suggests that the protein consists of two domains: (a) an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear ribonucleoprotein complex, and (b) a carboxyl-terminal portion that is homologous to beta-D-galactoside-specific lectins isolated from a number of animal tissues. This two-domain motif is reminiscent of several DNA- and RNA-binding proteins.

Amino Acid Sequence↗
PubMed1987

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin.

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.

Amino Acid Sequence↗