Isolation of a cDNA clone for the dihydrolipoamide acetyltransferase component of the human liver pyruvate dehydrogenase complex

Dihydrolipoamide acetyltransferase (E2) forms the structural core of pyruvate dehydrogenase complex. A cDNA clone (lambda E2-1) for mammalian E2 was identified from a human liver lambda gt11 library using anti-E2 serum. Affinity-selected antibodies using the fusion protein from lambda E2-1 immuno-reacted specifically with E2 of purified pyruvate dehydrogenase complex on immuno-blot analysis. The cDNA insert was approximately 2.3 kb in length with an internal EcoR1 site generating 1.4 and 0.9 kb fragments. A synthetic 17-mer oligodeoxynucleotide mixture based on the amino acid sequence surrounding the lipoic acid-containing lysine residue in bovine kidney E2 hybridized with the 2.3 kb cDNA insert and the 1.4 kb fragment.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential expression of the "B" subunit of the vacuolar H(+)-ATPase in bovine tissues.

The B subunit is one of two nucleotide-binding polypeptides found in all members of the vacuolar class of H(+)-translocating ATPases. We have isolated aDNA clone encoding the bovine brain B (58 kDa) subunit and have deduced its amino acid sequence. The bovine brain amino acid sequence is 99% identical to a partial cDNA reported from human brain. Northern blot analysis of RNA isolated from bovine tissues and a bovine kidney cell line reveals that two messages of approximately 3.2 and 2.0 kilobases (kb) are expressed in all tissues examined except brain, where only the 3.2-kb message can be detected. Northern blotting of RNA isolated from human fibroblast and human lung tumor cell lines reveals that three messages of approximately 6.0, 3.2, and 2.0 kb are expressed, whereas only the 3.2-kb message is expressed in a human brain tumor cell line. This is the first demonstration of tissue-specific expression of multiple forms of a vacuolar H(+)-ATPase subunit. We have also isolated a partial cDNA clone from bovine brain which appears to encode an isoform of the B subunit. The deduced amino acid sequence is 82% identical to the major bovine brain B subunit sequence; it does not hybridize with either the 3.2- or 2.0-kb message on Northern blot. Southern blot analysis of bovine genomic DNA with probes derived from both isolated cDNAs indicates that the bovine B subunit is encoded by a multigene family.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Isolation, characterization and chromosomal localization of cDNA clones for the E1 beta subunit of the pyruvate dehydrogenase complex.

A full-length cDNA clone for the E1 beta subunit of the human pyruvate dehydrogenase (PyrDH) complex was isolated from a human skin fibroblast cDNA library. When sequenced, it showed differences from the nucleotide sequence already published [Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. (1988) Proc. Natl Acad. Sci. USA 85, 41-45], such that 19 amino acids were different in the translated open reading frame. Northern blotting of human fibroblast cell lines revealed a major mRNA species of 1.6 kb and a weaker band of 5.5 kb. In a series of nine PyrDH-complex-deficient cell lines from patients with this deficiency, no patients had severely reduced amounts of mRNA, but there was one patient cell line with an increased amount of abnormal-size mRNA. Chromosome localization carried out with DNA blots from man-mouse hybrid cell lines indicated that the E1 beta subunit of pyruvate dehydrogenase is located on chromosome 3. A motif AXGXXXXGL(R/K)X15(D/E)Q was found in common with a variety of other oxo-acid oxidoreductases, but its function is not known.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs

The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.     

Isolation and characterization of a complementary DNA for the nuclear-coded precursor of the beta-subunit of bovine mitochondrial F1-ATPase

We have isolated a cDNA clone encoding the precursor of the beta-subunit of the bovine heart mitochondrial F1-ATPase. Two probes were used to isolate this precursor from a bovine heart cDNA library. One probe was a mixed-sequence oligonucleotide directed against a portion of the amino acid sequence of the mature protein, and the other probe was the F1-ATPase beta-subunit gene from Saccharomyces cerevisiae. Determination of the nucleotide sequence of this cDNA reveals that it contains a 1584-nucleotide-long open reading frame that encodes the complete mature beta-subunit protein and a 48 amino acid long NH2-terminal extension. This amino-terminal presequence contains four basic arginine residues, one acidic glutamic acid residue, four polar uncharged serine residues, and five proline residues. Southern blot hybridization analyses suggest that the bovine F1-ATPase beta-subunit precursor is encoded by a single genetic locus. RNA blot hybridization analyses reveal a single mRNA species of approximately 1.9 kilobases from both bovine liver and heart.     

Human elongation factor 1 beta: cDNA and derived amino acid sequence.

From a cDNA library in lambda gt11 derived from poly (A+)RNA of human ovarian granulosa cells a cDNA clone lambda HGP34, containing an EcoRI insert of 829 bp, was identified. After subcloning of the insert into pUC18, the clone pHGP34 was obtained and sequenced. The derived amino acid sequence, corresponding to a protein of 225 amino acids, shows a high degree of homology to elongation factor 1 beta (EF-1 beta) of Artemia salina (57%) and known peptide sequences of Xenopus laevis EF-1 beta (86%). We therefore assume that the protein coded for by pHGP34 represents human EF-1 beta. Northern analysis reveals an EF-1 beta specific mRNA of 900 bp. Southern analysis indicates that EF-1 beta in the human genome, like EF-1 alpha, appears to be specified by more than one gene. A high degree of sequence homology for EF-1 beta specific sequences is observed for bovine, rat and mouse species.     

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone

Summary We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3 untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results rere obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.