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.     

Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells

Sulfated glycoprotein 2 (SGP-2) is the major protein secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-2 is synthesized as a cotranslationally glycosylated 64-kDa precursor that is modified to a negatively charged 73-kDa form before intracellular cleavage to the mature 47- and 34-kDa subunits. A plasmid cDNA library was constructed from immunopurified mRNA, and a recombinant clone containing the entire protein coding sequence of SGP-2 was isolated. The 1857-nucleotide cDNA consists of a 297-nucleotide 5' noncoding segment, a 1341-nucleotide coding segment, and a 219-nucleotide 3' noncoding sequence. The 5' noncoding region contains five ATG codons followed by four short open reading frames. The derived SGP-2 sequence has a molecular weight of 51,379 and contains six potential N-glycosylation sites. Proteolytic processing sites for the preproprotein were determined by amino-terminal sequencing of the isolated SGP-2 subunits. Northern blots show a wide tissue distribution for the 2.0-kb SGP-2 message, and computer sequence analysis indicates a significant relationship between SGP-2 and human apolipoprotein A-I.     

Molecular cloning of porcine alpha 1-microglobulin/HI-30 reveals developmental and tissue-specific expression of two variant messenger ribonucleic acids.

A 1008 basepair (bp) cDNA clone encoding 335 amino acids followed by an inframe TGA translation termination codon and a 295-nucleotide 3' untranslated (UT) region has been isolated from a pig liver cDNA library. Based on the deduced amino acid and nucleotide sequence homology to a human cDNA (Kaumeyer, J.F., Polazzi, J.O. and Kotick, M.P. (1986) Nucleic Acids Res. 14, 7839-7850), the 5' amino terminus was found to code for alpha 1-microglobulin (alpha 1-M), a 183 amino acid protein belonging to the lipocalin protein superfamily (Pervaiz, S. and Brew, K. (1985) Science 228, 335-337). The 3' half encoded HI-30 which constitutes the Kunitz-type proteinase inhibitory (L-chain) domain of porcine inter-alpha-trypsin inhibitor (I alpha TI). In Northern blot hybridization, this cDNA identified two equally abundant mRNA species of approx. 1.3 kb and 1.6 kb in length. However, a 125 bp cDNA probe derived from the 3' UT region of the cDNA hybridized only to the 1.6 kb mRNA. The differences observed in the 3' UT region of these mRNAs suggest the utilization of alternative polyadenylation signals or presence of unprocessed nuclear RNA. Densitometric scanning of Northern blots indicated that alpha 1-M/HI-30 mRNA levels were higher (5-8-fold) in fetal and neonatal liver compared to that of primiparous pigs. In contrast, the RNA levels did not change significantly during pregnancy. Dot blot analysis of RNA indicated liver to be the major site of alpha 1-M/HI-30 mRNA expression with lower levels observed in the stomach. The results suggest that modulation of alpha 1-M/HI-30 gene expression could play a role during porcine growth. Increased I alpha TI L-chain mRNA levels may be particularly important in fetal and neonatal development when regulation of the inflammatory response and protection of macromolecules from proteolytic degradation is vital to survival and sustained growth.     

Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase

We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP-dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

Differential expression of Atlantic salmon thyrotropin β subunit mRNA and its cDNA sequence

To study the regulation of the thyroid system, an Atlantic salmon Salmo salar cDNA clone was isolated for thyroid stimulating hormone (TSH) β subunit gene. A cDNA (866 bp) was isolated from an adult Atlantic salmon pituitary cDNA library, this clone was sequenced and shown to be highly conserved when compared to other teleost β TSH subunit sequences. The cDNA was used as a probe for Northern blot analysis of total pituitary RNA from the different life cycle stages of Atlantic salmon. Northern blot analysis demonstrated that β TSH mRNA is expressed at all life cycle stages studied, including parr, smolt, immature fish at sea and sexually mature male fish. Densitometry of Northern blots showed that sexually mature male salmon had low levels of salmon β TSH mRNA compared to non-mature fish. Stunts, fish performing poorly in salt water, were shown to have elevated levels of β TSH mRNA when compared to healthy fish.     

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.     

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.     

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.     

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 rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.     

Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.     

Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex

We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P. Sugrue. 1992. J. Cell Biol. 118:1477-1488). We suggest that the function of pinin is to pin intermediate filaments to the desmosome. Therefore, pinin may play a significant role in reinforcing the intermediate filament- desmosome complex. cDNA clones coding for pinin were identified, using degenerative oligonucleotide probes that were based on the internal amino acid sequence of pinin for the screening of a cDNA library. Immunoblotting of expressed recombinant proteins with the monoclonal 08L antibody localized the 08L epitope to the carboxyl end of the protein. Polyclonal antibodies directed against fusion proteins immunoidentified the 140-kD protein in tissue extracts. Immunofluorescence analysis, using the antifusion protein antibody, demonstrated pinin at lateral epithelial boundaries, which is consistent with desmosomal localization. The conceptual translation product of the cDNA clones contained three unique domains: (a) a serine- rich domain; (b) a glutamine-proline, glutamine-leucine repeat domain; and (c) an acidic domain rich in glutamic acid. Although the 3' end of the open reading frame of the clone for pinin showed near identity to a partial cDNA isolated for a pig neutrophil phosphoprotein (Bellavite, P., F. Bazzoni, et al. 1990. Biochem. Biophys. Res. Commun. 170:915- 922), the remaining sequence demonstrated little homology to known protein sequences. Northern blots of mRNA from chicken corneal epithelium, MDCK cells, and various human tissues indicated that pinin messages exhibit tissue-specific variation in size, ranging from 3.2 to 4.1 kb. Genomic Southern blots revealed the existence of one gene for pinin, suggesting alternative splicing of the mRNA. Expression of the full-length cDNA clones in human 293 cells and monkey COS-7 cells demonstrated that a 140-kD immunoreactive species on Western blots corresponded to pinin. Pinin cDNA transfected into the transformed 293 cells resulted in enhanced cell-cell adhesion. Immunofluorescence staining revealed that the expressed pinin protein was assembled to the lateral boundaries of the cells in contact, which is consistent with the staining pattern of pinin in epithelial cells.     

Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat

gamma-Glutamyl transpeptidase (GGT) is a glutathione-metabolizing enzyme that has been extensively studied in relation to hepatocarcinogenesis. Using a cDNA for rat kidney GGT as a probe, we have isolated a full-length cDNA for human GGT from a hepatoma cell-line library. Nucleotide sequence analysis of the clone revealed a 2326-bp insert that includes a 5'-untranslated region of 487 nucleotides (nt), an open reading frame (ORF) of 1707 nt, and a 3'-untranslated region of 132 nt. The ORF encodes a protein with an amino acid sequence that is highly similar to that of the rat GGT precursor peptide, with an overall identity of 79%. The cDNA clone was used to probe Northern blots of hepatoma and kidney RNA from both human and rat. In both species, the GGT mRNA is longer in hepatoma than in kidney. In addition, the human mRNAs were longer than their counterparts in the rat. None of three human hepatocellular carcinomas examined showed a marked elevation in GGT mRNA levels relative to surrounding liver tissue.