Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.     

A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (B?hni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.     

Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y)

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.     

Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain

We have purified a novel GTP-binding protein (G protein) with a Mr of about 24,000 to homogeneity from bovine brain membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In the present studies, we have isolated and sequenced the cDNA of this G protein from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences. The cDNA of the G protein has an open reading frame encoding a protein of 220 amino acids with a calculated Mr of 24,954. This G protein is designated as the smg-25A protein (smg p25A). The amino acid sequence deduced from the smg-25A cDNA contains the consensus sequences of GTP-binding and GTPase domains. smg p25A shares about 28 and 44% amino acid homology with the ras and ypt1 proteins, respectively. In addition to this cDNA, we have isolated two other homologous cDNAs encoding G proteins of 219 and 227 amino acids with calculated Mr values of 24,766 and 25,975, respectively. These G proteins are designated as the smg-25B and smg-25C proteins (smg p25B and smg p25C), respectively. The amino acid sequences deduced from the three smg-25 cDNAs are highly homologous with one another in the overall sequences except for C-terminal 32 amino acids. Moreover, three smg p25s have a consensus C-terminal sequence, Cys-X-Cys, which is different from the known C-terminal consensus sequences of the ras and ypt1 proteins, Cys-X-X-X and Cys-Cys, respectively. These results together with the biochemical properties of smg p25A described previously indicate that three smg p25s constitute a novel G protein family.     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.     

A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors.

Several cDNA clones encoding a 46-kDa collagen-binding glycoprotein (gp46) from rat skeletal myoblasts were isolated and sequenced. The cDNA encoded a 17-amino acid signal peptide and a 400-amino acid mature protein, containing three potential N-linked oligosaccharide attachment sites. The cDNA sequence of gp46 shows 93% identity in the coding region with J6, a retinoic acid-inducible gene coding for a protein of unknown function described from embryonal carcinoma F9 cells. The first 41 NH2-terminal amino acids of the predicted J6 sequence are, however, different from the gp46 sequence as a result of a 7-base pair insertion in the gp46 cDNA. In addition, the NH2-terminal amino acid sequence of hsp47, a collagen-binding protein found in chick embryo fibroblasts, shows 64% identity to gp46 over 36 residues. Interestingly, this alignment begins 10 residues inward from the first amino acid in the mature form of gp46. A significant sequence similarity was observed between gp46 and members of the serine protease inhibitor (serpin) family. Unlike other serpins, however, gp46 is both a heat shock and a collagen-binding protein and is localized to the lumen of the endoplasmic reticulum, as suggested by the presence of the RDEL sequence at the COOH terminus. This sequence is similar to other proposed endoplasmic reticulum retention signals.     

Internal quadruplication in the structure of human interstitial retinol-binding protein deduced from its cloned cDNA

Interstitial retinol-binding protein (IRBP) is a glycoprotein that shuttles retinoids between the retina and pigment epithelium and is secreted by the photoreceptor cells of the vertebrate eye. Human retina cDNA libraries in lambda gt10 were screened with a previously isolated human IRBP probe (H.4 IRBP), yielding five overlapping cDNA clones generating a 4223-base sequence. A 17-kilobase pair clone (HGL.3) isolated by screening a human genomic library in EMBL3 with H.4 IRBP yielded a 2.5-kilobase pair SstI fragment that overlapped the 5' end of the cDNA sequence by 329 nucleotide residues. An open reading frame encoded the N-terminal sequence of human IRBP and predicted a protein consisting of 1262 amino acids with a molecular mass of 136,600. Two putative N-linked glycosylation sites were identified. The translated sequence suggests that there is a 16-amino acid presumptive signal peptide rich in hydrophobic residues and with a high alpha-helix probability preceding the N terminus of the mature protein. The amino acid sequence of human IRBP could be aligned with 87% identity with the amino acid sequences of 31 peptides (605 residues) purified from a tryptic digest of bovine IRBP. The protein sequence of human IRBP contains four duplicated segments (302-310 residues in length) with 33-38% identity. From the degree of identity between the bovine and human sequences, it is possible that IRBP evolved by several gene duplications that occurred 600-800 million years ago, before the emergence of the vertebrates.     

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.     

Structure and expression of a cloned cDNA for mouse interferon-beta

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.     

Nucleotide sequence of a cDNA for the common alpha subunit of the bovine pituitary glycoprotein hormones. Conservation of nucleotides in the 3'-untranslated region of bovine and human pre-alpha subunit mRNAs

A cDNA clone for the pre-alpha subunit of the pituitary glycoprotein hormones has been isolated from a bovine pituitary cDNA library through the use of a pool of synthetic oligodeoxynucleotide probes. This clone, designated pB alpha, contains a 564-base pair insert which includes a portion of the signal sequence, the entire coding sequence of the mature protein, and 224 base pairs of the 3'-untranslated sequence. As expected, the nucleotide and amino acid sequence of the mature bovine alpha subunit was homologous to the sequences reported for humans and rodents, with the most extensive homology occurring between bovine and rodents (85-90%). However, a comparison of the 3'-untranslated regions of pre-alpha subunit mRNA from three different mammalian species indicated that in bovine and rat, or in human and rat, these sequences have rapidly diverged, yielding respective homologies of 21 and 36%. In contrast, the sequence homology observed between the 3'-untranslated regions of bovine and human was 79%, which approaches the level of homology shared by their coding sequences. Thus, the conservation of the 3'-untranslated sequence in bovine and human pre-alpha subunit mRNA may be an indication that this region is functionally significant in these two species.     

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.     

Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjected to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in lambda gt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxyl-terminal third is rich in alpha helices.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats

Polysialoglycoprotein (PSGP) of unfertilized eggs of rainbow trout (Salmo gairdneri) consists of tandem repeats (about 25) of a glycotridecapeptide, Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly (* denotes the attachment site of a polysialoglycan chain) (Kitajima, K., Inoue, Y., and Inoue, S. (1986) J. Biol. Chem. 261, 5262-5269). By using oligodeoxynucleotide probes based on the above sequence, we isolated a genomic clone for apoPSGP which contains 39-base pair repeats (5'-GACGACGCCACCTCTGAAGCT-GCGACCGGCCCGTCTGGC-3') encoding the tridecapeptide. Using a fragment of this genomic DNA as a probe, we next screened a cDNA library constructed with mRNA from immature ovaries of rainbow trout. Nucleotide sequencing analyses of cDNA clones thus obtained revealed that apoPSGP is encoded by multiple mRNA species consisting of diverged numbers (6-32) of the 39-base repeat encoding the tridecapeptide unit and homologous 5'- and 3'-bordering regions. The encoded protein consists of three distinct regions: the N-region consisting of a putative signal peptide and a pro-peptide, the R-region containing diverged numbers of the tandem repeat of 13-amino acid residues, and the C-region with six amino acid residues. Southern blot analysis showed that multiple mRNAs are transcribed from multiple genes for apoPSGP containing diverged numbers of the 39-base pair repeat. Thus, the genes for apoPSGP constitute a multigene family. Expression of the mRNAs is stage and organ specific, i.e. they are expressed only in immature ovaries and not in mature ovaries or in any other organ.     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.