Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.     

Endocytosis of N-acetylglucosamine-containing glycoproteins by rat fibroblasts expressing a single species of chicken liver glycoprotein receptor

A cDNA clone for the chicken liver receptor which mediates endocytosis of glycoproteins containing terminal N-acetylglucosamine has been isolated and sequenced, confirming the previously obtained amino acid sequence of this protein (which is also known as the chicken hepatic lectin). This cDNA was introduced into Rat-1 fibroblasts and expressed using the promotor in the long terminal repeat of Moloney murine leukemia virus. Cells expressing chicken receptor were identified by screening with antireceptor antibodies followed by fluorescein-conjugated second antibodies. Receptor expressed in these cells was indistinguishable on gel electrophoresis from receptor isolated from liver. Three clonally isolated lines were examined for their ability to bind agalacto-alpha 1-acid glycoproteins at 0 degrees C and to take up and degrade this ligand at 37 degrees C. The receptor number (50,000/cell), affinity for ligand (35 nM), and uptake rate (5 molecules ligand/surface receptor/h) are similar to those previously observed for chicken hepatocytes, and for the uptake of asialoglycoproteins by rat hepatocytes and hepatoma cells. These findings indicate that the chicken receptor correctly traverses the endocytic pathway in a rat cell even though the cytoplasmic domain of this protein shows no primary structural homology with the corresponding portion of the rat liver receptor or with receptors found in fibroblasts.     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

We have isolated a cDNA clone for chicken thioredoxin by differential screening of a cDNA library. The protein product which this clone encodes is very similar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Comparison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates structural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonproliferative tissue (brain) is much lower than in chicken embryo fibroblasts.     

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.     

Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.     

The expression of a functional cDNA encoding the chicken cytochrome P-450arom (aromatase) that catalyzes the formation of estrogen from androgen

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.     

Cloning and sequencing of a full-length rat sucrase-isomaltase-encoding

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to study cellular differentiation in the small intestine. We isolated a 6.1-kb SI cDNA clone (GC1.4) from a size-fractionated cDNA library from rat intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt) with an open reading frame (ORF) of 1841 amino acids (aa). The nt sequence correctly predicts several known aa stretches in the protein. The deduced aa sequence showed 78 and 75% overall identity with the rabbit and human SI, respectively. At the active sites of both S and I, the rat nt sequence encodes stretches of 14 and 16 aa, respectively, which show 100% identity to rabbit and human SI. In the region immediately beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additional 0-glycosylations of rat SI. The cDNA contains a 3'-UTR (untranslated region) of 499 nt with polyadenylation signal sequence and a poly(A) tract. The ATG start codon was found 41 nt downstream from the 5' end of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon. The results indicate that our cDNA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Molecular cloning and primary structure of rat alpha 1-antitrypsin

A cDNA clone encoding rat alpha 1-antitrypsin has been isolated from a lambda gt-11 rat liver cDNA library using an antigen-overlay immunoscreening method. The nucleotide sequence of this cDNA clone is 1306 base pairs in length and has a coding region of 1224 base pairs which can be translated into an alpha 1-antitrypsin precursor protein consisting of 408 amino acid residues. The cDNA sequence contains a termination codon, TAA, at position 1162 and a polyadenylation signal sequence, AATAAT, at position 1212. The calculated molecular weight of the translated mature protein is 43,700 with 387 amino acid residues; this differs from purified rat alpha 1-antitrypsin's apparent molecular weight of 54,000 because of glycosylation. Five potential glycosylation sites were identified on the basis of the cDNA sequence. The translated mature protein sequence from the cDNA clone matches completely with the N-terminal 33 amino acids of purified rat alpha 1-antitrypsin, which has an N-terminal Glu. The cDNA encoding rat alpha 1-antitrypsin shares 70% and 80% sequence identity with its human and mouse counterparts, respectively. The reactive center sequence of rat alpha 1-antitrypsin is highly conserved with respect to human alpha 1-antitrypsin, both having Met-Ser at the P1 and P1' residues. Genomic Southern blot analysis yielded a simple banding pattern, suggesting that the rat alpha 1-antitrypsin gene is single-copy. Northern blot analysis using the cDNA probe showed that rat alpha 1-antitrypsin is expressed at high levels in the liver and at low levels in the submandibular gland and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

Molecular cloning, cDNA structure and deduced amino acid sequence for the hormone-induced regulatory subunit (RII beta) of cAMP-dependent protein kinase from rat ovarian granulosa cells

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.     

The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenine-induced oocyte maturation.

Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat Gi-2 cDNA. The deduced amino acid sequence was 85% and 89% identical to rat Gi-2 and rat Gi-1, respectively. The alpha subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of Gi, suggesting that the cysteine residue is the site of ADP-ribosylation.