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.     

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.     

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.     

Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase.

cDNA clone for human liver S-adenosylmethionine synthetase (liver-specific isoenzyme) was isolated from a cDNA library of human liver poly(A)+ RNA. The cDNA sequence encoded a polypeptide consisting of 395 amino acid residues with a calculated molecular mass of 43675 Da. Alignment of the predicted amino acid sequence of this protein with that of rat liver S-adenosylmethionine synthetase showed a high degree of similarity. The coding region of the human liver S-adenosylmethionine synthetase cDNA sequence was 89% identical at the nucleotide level and 95% identical at the amino acid level to the sequence for rat liver S-adenosylmethionine synthetase.     

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots.

A cDNA clone obtained from developing peanut (Arachis hypogaea) seedling roots, when expressed in Escherichia coli and insect cells (Sf9) gave a 29 kDa subunit protein. The native recombinant protein agglutinates neuraminidase treated human erythrocytes and the agglutination is inhibited by galactose. Nucleotide sequence and predicted amino acid sequence analyses indicate that it is different from peanut seed (PNA and SGL) and nodule (NGLa and NGLb) galactose-binding lectins.     

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.     

Primary structure of the human fgr proto-oncogene product p55c-fgr.

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.     

Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of MACIF, an inhibitor of membrane channel formation of complement

Human erythrocytes contain a membrane protein, MACIF, which inhibits the formation of a membrane attack complex (MAC) of complement. We have cloned and sequenced the complementary DNA of MACIF messenger RNA. The amino acid sequence predicted from its nucleotide sequence consists of 128 amino acids. The amino-terminal 25 residues may correspond to a signal peptide. The carboxy-terminal sequence confirmed that MACIF is a glycosylphosphatidylinositol (GPI)-anchored protein. The amino acid sequence of MACIF was partially determined by established techniques for protein chemistry and the resultant sequence was consistent with that predicted from the nucleotide sequence. The results of sequence analyses also suggested that asparagine at the 18th position was N-glycosylated. When mRNA obtained from the MACIF cDNA clone with SP6 RNA polymerase was microinjected into Xenopus oocytes, the oocytes synthesized a product which exhibited MACIF activity and reacted with anti-MACIF antibody. Comparison of the predicted sequence revealed significant homology with mouse Ly-6 antigens.     

A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase

While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.     

The complete primary structure of the human snRNP E protein.

The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.     

cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Identification from cDNA of the precursor form of a chondroitin sulfate proteoglycan core protein

The yolk sac carcinoma cell line L2 secretes a chondroitin/dermatan sulfate proteoglycan that has an Mr 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1321-1325). From additional cDNA sequences described in this report we have identified the prepro core protein precursor of the yolk sac carcinoma chondroitin/dermatan sulfate proteoglycan. From the amino acid sequence of the core protein precursor can be deduced the protein processing events in the biosynthesis of the proteoglycan. The amino acid sequence shows that the 104-amino acid mature core protein is processed from a 179-amino acid prepro core protein precursor which, in addition to the mature core protein, contains a 26-amino acid signal peptide as well as a 49-amino acid propeptide. The molecular weight of the prepro core protein predicted from the cDNA sequence (Mr = 18,600) was in good agreement with the molecular weight of the in vitro translation product (Mr = 19,000) of hybrid-selected mRNA. Accordingly, we have designated the proteoglycan core protein PG19. Further analysis of the PG19 mRNA by RNA sequencing confirmed the identification of the core protein translation initiation codon by revealing stop codons in all three reading frames of the upstream mRNA sequence. Primer extension analyses demonstrated that the 5' untranslated sequence of the proteoglycan mRNA is approximately 220 nucleotides in length, which, combined with the length of cDNA clones, accounts for the entire length of the coding sequence of PG19 mRNA from L2 cells. The cDNA sequences presented here establish the complete protein sequence of PG19 and provide evidence of polypeptide processing during the biosynthesis of the proteoglycan core protein.     

Cloning,characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11

A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.     

Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system.

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3' non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.