Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein S11

A cDNA clone specific for rat ribosomal protein S11 was isolated by hybrid-selected translation from the cDNA library made for 8-9 S poly(A) RNA from regenerating rat liver. Since this cDNA had not enough length, another clone was selected by colony hybridization using a fragment of isolated cDNA as a probe. The nucleotide sequence of the cDNA was determined. The sequence contains 2 base pairs from the 5' noncoding region, the entire coding region of 477 base pairs, and the 3' noncoding region of 55 base pairs besides the poly(A) tail. The primary structure of the protein S11 was deduced from the nucleotide sequence. It consists of 157 amino acids. Its molecular weight is 18,299. The calculated amino acid composition is consistent with the reported composition of S11 determined on the protein hydrolysate. The amino acid sequence showed a marked homology with that of S16 of Halobacterium cutirubrum and an appreciable homology with that of S17 of Escherichia coli.     

Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker

The mAb KS1/4 recognizes a novel cell surface glycoprotein on a variety of epithelial carcinomas which may be a useful target Ag for antibody-directed diagnostic and therapeutic approaches. Here we report the isolation and characterization of a full length cDNA clone coding for the KS1/4 Ag, as well as, physical and biochemical studies on the antigen derived from an adenocarcinoma of the lung cell line. Affinity purification of the KS1/4 Ag reveals three glycosylated species by NaDodSO4 PAGE with molecular weights of 42, 40, and 35 kDa. The 42- and 40-kDa species are similar at the protein level, differing by their degree of glycosylation, and the 35-kDa protein results from a dibasic proteolytic cleavage of the larger m.w. species. Although both the 42- and 40-kDa forms are found on the cell surface, the 40-kDa protein appears to be the predominant species. A cDNA clone containing the complete KS1/4 coding sequence and the 5'- and 3'-non-translated regions was isolated from a library constructed from the human adenocarcinoma of the lung derived cell line, UCLA-P3. The cDNA clone contains an open reading frame of 314 amino acids which includes a putative signal sequence of 23 amino acids. Northern blot analysis shows a single RNA species of 1.5-kb. Sequence analysis of the 5' and 3' noncoding regions of the KS1/4 cDNA revealed homologies to known proto-oncogenes and inflammatory mediators.     

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.     

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Guinea-pig casein A cDNA. Nucleotide sequence analysis and comparison of the deduced protein sequence with that of bovine alpha s2 casein

The nucleotide sequence (1036 bases) of guinea-pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5' noncoding region, and the complete coding and 3' noncoding region. The remaining 5' noncoding sequence was obtained by primer extension. The deduced 223-amino-acid-coding sequence of guinea-pig pre-casein A exhibited 30% homology with bovine alpha s2 casein, the most striking similarities being in the locations of potential phosphorylation sites.     

Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.     

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Nucleotide sequence of the gene encoding the serotype-specific glycoprotein of UK bovine rotavirus

The nucleotide sequence of the gene which encodes the major outer-shell glycoprotein of UK bovine rotavirus has been determined. The dsRNA genome segment encoding this protein was converted into ds cDNA and cloned into pBR322 for sequence studies. The gene is 1062 base pairs in length and contains a single, long, open reading-frame capable of coding for a protein of 326 amino-acids. This would leave 5' and 3' non-coding regions of 48 and 36 nucleotides in the mRNA. The predicted amino-acid sequence contains three possible glycosylation sites of the type Asn-X-Ser Thr, and an extremely hydrophobic N-terminal region. This sequence is discussed in the light of the known properties and functions of the protein.     

The primary structure of the 32-kDa subunit of human replication protein A

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.     

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5'' exons and possible mechanism for the genesis of the 3'' end of v-src.

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.