Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Gene expression and cDNA cloning identified a major basic protein constituent of bovine seminal plasma as bovine monocyte-chemoattractant protein-1 (MCP-1).

P6 is one of the major basic proteins of bovine seminal plasma. Using cell-free translation of poly(A)+RNA from bovine seminal vesicle tissue and monospecific anti-P6-IgGs, we show that P6 is a secretory product of the seminal vesicles. Immunohistochemical experiments supported this finding. Immunoscreening of a lambda gt11 cDNA library derived from seminal vesicle poly(A)+RNA furnished a number of positive cDNA clones, from which clone pH42 was characterized by sequencing. The partial amino acid sequence of a CNBr-fragment of P6 permitted identification of the reading frame of clone pH42 encoding the precursor protein of P6. The P6 precursor contains a signal peptide of 23 amino acids followed by the mature P6 sequence of 76 amino acid residues. The cDNA sequence of pH42 was 80% homologous with that of the human monocyte-chemoattractant protein-1 (hMCP-1). The respective amino acid sequences for the precursor molecules are 72% identical. Northern analysis of seminal vesicle poly(A)+RNA using pH42 as probe probe identified a 0.9-kb P6 mRNA. Stimulation of P6 mRNA expression by phytohemagglutinin in bovine peripheral mononuclear leukocytes suggests that P6 is identical to bovine MCP-1.     

cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.     

Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck

In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II)

Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.     

Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3-bisphosphoglycerate synthase

We have isolated and sequenced a cDNA clone containing the entire coding region of rabbit reticulocyte 2,3-bisphosphoglycerate (DPG) synthase. The cDNA was verified by translation of the hybridization-selected RNA and by demonstrating identity of the deduced amino acid (aa) sequence to the sequences of CNBr peptides of the purified enzyme. The aa sequence of the enzyme was homologous to the reported sequence of the human enzyme [Haggarty et al., EMBO J. 2 (1983) 1213-1220], especially in the N-terminal half (aa 1-142). Northern blot analysis of rabbit reticulocyte poly(A)+ RNA revealed a single species of mRNA with about 1700 nucleotides.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

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.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of a pollen-specific cDNA clone from Zea mays and its expression.

A pollen-specific cDNA clone, Zmc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.     

Structure of a bacterial photosynthetic membrane: integrity of reaction centers following proteolysis and detergent solubilization

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were previously demonstrated not to be expressed. A plasmid cDNA library consisting of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32p-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/acidic residues being 1.63.