Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta- spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.     

Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin.

A cDNA clone for nonerythroid alpha-spectrin was identified by direct immunological screening of a chicken smooth muscle cDNA library. A library prepared in the expression plasmids pUC8 and pUC9 was screened with an antiserum specific for chicken alpha-spectrin. Blots of poly(A)+ RNA from various tissues of chicken and mouse show that the cDNA hybridizes to an 8-kilobase mRNA. The cDNA hybridizes to a single-copy sequence on Southern blots of chicken genomic DNA. The complete nucleic acid sequence of the clone has a single 1419-base open reading frame. The derived amino acid sequence is organized into two partial and three complete 106-amino-acid repeats that show homology to the repeats described for human erythroid alpha- and beta-spectrin. Immunological and biochemical data indicate that chicken nonerythroid and human erythroid alpha-spectrin are two of the more widely diverged members of the spectrin family of proteins. In this respect, the degree of homology found between them was unexpected. Our data suggest a common evolutionary origin for these two alpha-spectrins and allow some predictions concerning spectrin gene structure.     

Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man.

The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonucleotide based on the bovine cDNA sequence.     

Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase.

We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in lambda Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxyl-terminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.     

Molecular cloning of a cDNA for androgen-regulated proteins secreted by the mouse epididymis

A cDNA clone encoding androgen-dependent proteins secreted by the mouse epididymis was isolated by screening a cDNA library using differential hybridization, according to the selective expression of the mRNA in the normal but not in the castrated mouse. Translation of mRNA hybrid-selected by this 1.4 kb clone (M53) yielded proteins of Mr 26,000 which were processed in vitro in the presence of microsomal membranes into proteins of Mr 24,000. Northern blot analysis of epididymal total RNA revealed at least two populations of mRNA (1.4 and 1.8 kb) homologous to the M53 clone, which were restricted to the caput epididymidis. Studies in vivo demonstrated that testosterone regulates the concentration of these mRNA populations. Analysis of epididymal total RNA from ten individual animals provided no evidence that the M53 mRNA populations are the products of allelic variants of a gene. Southern analysis of mouse genomic DNA revealed single bands with most of the tested restriction enzymes. Furthermore, cross-hybridization to the M53 cDNA revealed homologous mRNA species in rat, human, rabbit, ram and boar epididymal RNA.     

Molecular cloning, sequence analysis and expression of putative chicken insulin-like growth factor-I cDNAs

A cDNA library was constructed from mRNA isolated from the liver of a 5-week-old female broiler chicken; at this age the level of insulin-like growth factor-I (IGF-I) mRNA was expected to be high. Three clones, of sizes 2.3, 0.86 and 0.2 kb, were isolated by using a homologous human (IGF-I) probe. The DNA sequence of these clones has been determined and the potential amino acid sequence deduced. The sequence of the mature chicken IGF-I peptide shows a high degree of homology with IGF-I from other species, providing evidence for the identity of these clones. Alternative splicing of the chicken IGF-I mRNA has been found in the region potentially encoding the leader peptide. This may give rise to two forms of prepeptide, differing in the length and nature of their leader peptide. The 0.86 kb cDNA has been used as a probe to Northern blots of chicken mRNA. A major band of approximately 0.65-0.85 kb was seen, plus several minor bands of larger molecular weights. Analysis of genomic Southern blots shows that there is one copy of the chicken IGF-I gene.     

Salmonella serotype Typhimurium infection of bovine Peyer's patches down-regulates plasma membrane calcium-transporting ATPase expression

To identify host genes differentially expressed during Salmonella enterica serotype Typhimurium infection, an RNA differential display was made with total RNA extracted from ileal loops that were infected with Salmonella Typhimurium 2.5 h after infection. Down-regulated cDNA was identified in bovine Peyer's patches after infection that was highly homologous to a human plasma membrane calcium-transporting ATPase (PMCA). Differential expression of PMCA, evaluated by Northern analysis, was found to have more than a 4.6-fold decrease in expression of mRNA (size, approximately 5.1 kb). PMCA mRNA was detected by in situ hybridization exclusively within epithelial cells in the Peyer's patches. cDNA (4.4 kb) was amplified by rapid amplification of cDNA ends, cloned, and sequenced and showed a high homology to hPMCA. Bovine PMCA is down-regulated in epithelial cells of Peyer's patches after infection with Salmonella Typhimurium and, subsequently, may influence cellular calcium levels that contribute to the inflammatory processes in the pathogenesis of diarrhea.     

Activin receptor mRNA expression in rat testicular cell types.

cDNA encoding the extracellular domain of the rat activin receptor was cloned using the polymerase chain reaction (PCR). This cDNA is highly homologous to cDNA encoding the extracellular domain of the mouse activin receptor, whereas at the protein level the extracellular domains of both receptors are identical. Employing this cDNA as a probe in Northern blot analysis, expression of two activin receptor mRNAs (6 kb and 4 kb) was observed, in testes of immature and mature rats. Between day 21 and 28 of postnatal development, a large increase in testicular expression of the 4 kb mRNA was found, suggesting expression of this activin receptor mRNA in germ cells. The 4 kb mRNA was indeed present in isolated pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. Sertoli cells obtained from immature and mature rats expressed both the 6 kb and 4 kb mRNAs, whereas the expression of these mRNAs in Leydig cell preparations was very low. These results may imply that activin has multiple actions in the control of testicular function.     

cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns.     

Cloning and expression of cDNA for human poly(ADP-ribose) polymerase.

cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.     

cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.

The antiinflammatory glucocorticoids are potent inhibitors of cyclooxygenase, a key regulator of prostaglandin synthesis; yet, the mechanism(s) by which this occurs is not fully understood. We have cloned a 4.1-kilobase (kb) cDNA, distinct from the previously cloned cyclooxygenase (2.8 kb), that confers cyclooxygenase activity to transfected cells. The mRNA for this newly discovered cyclooxygenase is unique for its long 3' untranslated region containing many AUUUA repeats. Levels of the 4.1-kb cyclooxygenase mRNA are rapidly increased by serum or interleukin 1 beta in mouse fibroblasts and human monocytes, respectively, and decreased by glucocorticoids, whereas levels of the 2.8-kb cyclooxygenase mRNA do not change. Similar effects are seen in the presence of cycloheximide where the 4.1-kb, but not the 2.8-kb, mRNA is greatly superinduced. Thus, there are both constitutive (2.8 kb) and regulated (4.1 kb) cyclooxygenase species, the latter most likely being a major mediator of inflammation.