Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.     

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).     

Mouse ornithine decarboxylase. Complete amino acid sequence deduced from cDNA

cDNA containing the full coding region of mouse ornithine decarboxylase was isolated. The complete nucleotide sequence of the cDNA was determined by the dideoxy method, and the amino acid sequence of ornithine decarboxylase was thereby deduced. The protein contains 461 amino acids and has a molecular weight of 51,172. The isoelectric point is predicted from the deduced amino acid sequence to be 5.1. On the basis of its amino acid sequence, the protein is predicted to be comprised predominantly of alternating domains of alpha-helix and beta-sheet.     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence

Alcohol dehydrogenase (ADH) catalyzes the rate-determining reaction in the metabolism of ethanol. We report here the complete nucleotide sequence of a cDNA encoding rat liver ADH, and the deduced amino acid (aa) sequence of the protein. The rat enzyme contains a cluster of aa substitutions and an aa insertion in the region between aa residues 111 and 118, which is near the intron-exon junction reported for the human ADH gene. It also contains an additional cysteine in the highly variable region from aa residues 108-125 which may account for the unusual lability of rat ADH compared with ADH from other species.     

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.     

Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.     

Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.     

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.     

An amino acid sequence common to both cartilage proteoglycan and link protein

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. In the course of determining the primary structure of link protein, two peptides produced by digestion of rat chondrosarcoma link protein with trypsin or chymotrypsin have been selectively purified by immunoaffinity chromatography on a column of monoclonal anti-link protein antibody (8A4) immobilized to Sepharose 4B. These peptides have been sequenced using the double-coupling dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate procedure. A consensus sequence, Cys-X-Ala-Gly-Trp-Leu-X-Asp-Gly-Ser-Val-X-Tyr-Pro-Ile-X-X-Pro, obtained by comparing the affinity-isolated tryptic peptide with the affinity-isolated chymotryptic peptide and an overlapping tryptic peptide, shows homology with a sequence obtained from the NH2-terminal of a CNBr peptide from proteo glycan core protein of bovine nasal cartilage: Ser-Ser-Ala-Gly-Trp-Leu-Ala-Asp-Arg-Ser-Val-Arg-Tyr-Pro-Ile-Ser-. We suggest that the common sequence is structurally important to the function of these proteins and may be involved in the binding of both link protein and proteoglycan to hyaluronic acid.     

cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequence.

A FKBP cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.