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PubMed2001

Comparison of sequences of cDNA clones obtained from oligo-capping cDNA libraries with those from unigene.

We compared in detail the characteristics of the sequences of the cDNA clones obtained by the oligo-capping method (oligo-capping clones) with that of the sequences in the UniGene database. To compare the completeness of the sequences, three new variables, "fullness-proportion of clones" (the ratio of complete clones to total clones in a library), "fullness-proportion of genes" (the ratio of complete genes to total genes in a library), and "fullness-proportion of database" (the ratio of complete genes to total genes in a database sampled from a library), were defined. The fullness-proportion of clones of oligo-capping clones was 57.3%, 2.2 times larger than that of UniGene (25.9%). The fullness-proportion of genes of oligo-capping clones was 41.8%, 2.4 times larger than that of UniGene (17.8%). When gene length was restricted to > or = 1.5 kb, the fullness-proportion of genes of oligo-capping clones was four times larger than that of UniGene. The fullness-proportion of database of oligo-capping clones was approximately the same as that of UniGene. By simulating the clone redundancy, this coincidence was found to be due to the large redundancy of the UniGene database. Consequently, the cDNA sequence database of oligo-capping clones enabled high throughput selection of full-length cDNA clones.

Algorithms↗
PubMed2000

Characterization of zebrafish full-length prothrombin cDNA and linkage group mapping.

In this paper, we report the complete cDNA sequence of zebrafish prothrombin. The cDNA sequence predicts that zebrafish prothrombin is synthesized as a pre-proprotein consisting of a Gla domain, two kringle domains, and a two-chain protease domain. Zebrafish prothrombin is structurally very similar to human and other vertebrate prothrombins. Zebrafish and human prothrombin share 53% amino acid identity whereas zebrafish and hagfish prothrombin share 51% identity. Amino acid alignments of various prothrombins identified conservation of many of the functional/structural motifs suggesting that the vertebrate prothrombins may have similar functions. The three-dimensional structure of prothrombin predicted by homology modeling also revealed that the prothrombin fragment 1 and the catalytic domain structures are well conserved except for the insertion of an extra 7-amino-acid loop in the connecting region (CR) between the Gla and kringle I domain of fragment 1. Linkage analysis revealed that the prothrombin gene locus on linkage group 7 in zebrafish is syntenic to the human chromosome 11-prothrombin region suggesting its preservation through evolution. The availability of this cDNA sequence in zebrafish adds to our knowledge of the zebrafish hemostatic system and provides support for the view that similarities between zebrafish and mammalian coagulation exist, thus underscoring the relevance of the zebrafish model for studying human hemostasis.

Amino Acid Sequence↗
PubMed1991

The complete amino acid sequence of the human transglutaminase K enzyme deduced from the nucleic acid sequences of cDNA clones.

In order to study the expression and role of transglutaminases in the formation of the cross-linked cell envelope of human epidermis, we have used a synthetic oligonucleotide encoding the consensual active site sequence of known transglutaminase sequences. By Northern blot analysis, newborn foreskin epidermis expresses three different mRNA species of about 3.7, 3.3, and 2.9 kilobases while normal cultured epidermal keratinocytes express only the 3.7- and 2.9-kilobase species. The largest species corresponds to a known ubiquitous tissue type II or transglutaminase C activity, the smallest corresponds to a known type I or transglutaminase K activity, and the mid-sized component apparently encodes a transglutaminase E activity that has recently been shown to be expressed in terminally differentiating epidermis (Kim, H. C., Lewis, M. S., Gorman, J. L., Park, S. C., Girard, J. E., Folk, J. E. & Chung, S. I. (1990) J. Biol. Chem., in press). Using the active site oligonucleotide as a probe, we have isolated and sequenced cDNA clones encoding the transglutaminase K enzyme. The deduced complete protein sequence has 813-amino acid residues of 89.3 kDa, has a pl of 5.7, and is likely to be an essentially globular protein, which are properties expected from the partially purified enzyme. It shares 49-53% sequence homology with the other transglutaminases of known sequence, especially in regions carboxyl-terminal to the active site, and possesses sequences likely to confer its Ca2+ dependence. Interestingly, its larger size is due to extended sequences on its amino and carboxyl termini, absent on the other transglutaminases, that may define its unique properties.

Amino Acid Sequence↗
PubMed2006

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus.

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.

Amino Acid Sequence↗
PubMed1997

Sequencing of cDNA encoding serum albumin and its extrahepatic synthesis in the Mongolian gerbil, Meriones unguiculatus.

We have sequenced serum albumin cDNA from liver of the Mongolian gerbil, Meriones unguiculatus. The deduced amino acid sequence showed 82.6% and 73.6% identity with the corresponding proteins from rats and humans, respectively. Identical cDNA was detected in pancreas by reverse transcription followed by polymerase chain reaction (RT-PCR). Further amplification of cDNA by nested PCR revealed the presence of the same cDNA in the brain and kidney. These results indicate that serum albumin is expressed in some extrahepatic tissues. In rats, an albumin-related 70-kDa protein (P70) has been proposed to be associated with cobalt-induced epilepsy (Onozuka et al. (1995) Neurochem. Res., 20, 901-905). We intensively searched for a P70-like protein in the brain of an epilepsy-prone gerbil strain, MGS/Idr, by the RT-PCR and nested PCR using several pairs of primers based on the albumin cDNA sequence. However, we found only mRNA for albumin itself.

Amino Acid Sequence↗
PubMed1988

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones.

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).

Amino Acid Sequence↗
PubMed1990

A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis.

A cDNA clone isolated from a lambda gt11 expression library of teratocarcinoma OTT6050 specifies for a glycoprotein with a molecular weight of about 44,000. The new glycoprotein was termed heparin binding protein-44 (HBP-44), since it was absorbed to a heparin-agarose column and was eluted from it by a buffer containing 1.5 M NaCl. HBP-44 mRNA was intensely expressed in PYS-2 parietal endoderm cells and in the kidney, and the RNA level increased about 10-fold during differentiation of F9 embryonal carcinoma cells to parietal endoderm cells. From the cDNA sequence, HBP-44 was concluded to be rich in charged amino acids, and large segments of the protein appeared to form alpha-helixes. The protein was considered to be anchored to the membrane by a cluster of hydrophobic amino acids present in the N-terminal region. Indeed, the N-terminal sequence of HBP-44 was homologous to asialoglycoprotein receptor, which is anchored to the membrane by the N-terminal region. Furthermore, a portion of the N-terminal region of HBP-44 was homologous to the leucine zipper domain. Except for the N-terminal region, HBP-44 had over-all homology with structural proteins such as myosin heavy chain. We propose that HBP-44 is extruded from plasma membranes and interacts with heparin and related molecules and that it is involved in the interactions of plasma membranes with basement membranes.

Amino Acid Sequence↗
PubMed1987

An mRNA encoding poly-ubiquitin in porcine corpus luteum: identification by cDNA cloning and sequencing.

A cDNA library derived from a particular fraction of mRNA of porcine corpus luteum was screened by hybrid-release. cDNA clone pCL208 selected an mRNA which, in cell-free translation, directed the synthesis of a major polypeptide of 28 kD. Sequence analysis of clone pCL208 revealed that the entire coding sequence contained three complete repeats and a fourth incomplete repeat of porcine ubiquitin, joined head-to-tail without any spacer peptide sequences. The ultimate ubiquitin molecule contained a Phe residue as a carboxy-terminal extension. The coding sequence terminated with the stop codon TAA followed by a short untranslated region, the polyadenylation signal AATAAA, and a tract of A residues. Northern analysis indicated the presence of two types of ubiquitin-specific mRNAs, i.e., 1100 and 2500 bp, in porcine corpus luteum.

Amino Acid Sequence↗
PubMed1995

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Amino Acid Sequence↗
PubMed1996

Slim defines a novel family of LIM-proteins expressed in skeletal muscle.

We have assembled the complete protein sequence of the skeletal muscle LIM-protein SLIM by aligning overlapping cDNA sequences. These cDNA sequences were identified from our own sequencing and from BLASTn searches of non-redundant cDNA databases. The predicted SLIM protein sequence included four LIM-domains and a novel single zinc finger domain located in the N-terminal region. Similar sequences to SLIM were identified and termed SLIM2 and SLIM3. The SLIM3 cDNA sequence was identified subsequently as a partial sequence of the of the LIM-protein DRAL. The number and spacing of the LIM domains was common to all three protein sequences. The mRNA for each protein was detected in human masseter muscle RNA by Northern analysis. We suggest that these proteins belong to a novel family of LIM proteins that are expressed in human skeletal muscle.

Amino Acid Sequence↗
PubMed2002

cDNA hox sequences 3' of the homeobox isolated from the sea urchin Holopneustes purpurescens are definitive for sea urchin Hox orthologues.

In an investigation into Hox genes in sea urchin development, cDNA nucleotide sequences were isolated from seven Hox genes of Holopneustes purpurescens by 3' RACE. Each sequence extended from the homeobox through the coding region 3' of the homeobox to the 3'untranslated region and, in the majority of sequences, to a poly(A) signal and a poly(A) tail. The cDNA sequences 3' of the homeoboxes were very different between the seven Hox genes. In contrast, the sequences within each Hox gene 3' of the homeobox were very similar, especially at the amino acid level, between the sea urchin species for which sequences are available. The coding regions 3' of the homeodomains are thus a means of unambiguously identifying homologies between sea urchin Hox genes. A phylogenetic analysis where amino acid motifs in these 3' coding regions were aligned with similar motifs in chordate species led us to classify two posterior sea urchin Hox genes, of uncertain affinity, as orthologues of Hox9 and Hox10 genes. Putative cytoplasmic polyadenylation elements were mapped in the 3' untranslated region of sea urchin Hox genes.

3' Untranslated Regions↗
PubMed1991

Cloning and sequencing of cDNA coding for rabbit alpha-1-antiproteinase F: amino acid sequence comparison of alpha-1-antiproteinases of six mammals.

Rabbit liver cDNA coding for alpha-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit alpha-1-antiproteinase F showed 74 and 64% homology to human alpha-1-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of alpha-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.

Amino Acid Sequence↗
PubMed1981

Plasmacytomas with more than one immunoglobulin kappa mRNA: implications for allelic exclusion.

Although only one allele of an immunoglobulin gene is thought to be expressed as a polypeptide by a given lymphocyte ("allelic exclusion"), three murine plasmacytomas were found to contain more than one kappa light chain mRNA species, as evidenced by the sequences of distinct kappa cDNA clones. Two different kappa cDNA sequences were cloned from BFPC 61 microsomal mRNA, two from MOPC 173, and three from S107. One cDNA sequence from each tumor matches the known secreted polypeptide, while the variant sequences differ in the variable (V) region. Hence fusion of a V kappa gene to a joining (J kappa) gene has occurred independently on separate homologous chromosomes and each allele is transcriptionally competent. The BFPC 61 variant sequence contained a normal V kappa sequence linked out of phase to J kappa 2; hence allelic exclusion in this line is accounted for by an error in DNA rearrangement. One S107 variant cDNA has an untranslatable sequence linked to J kappa--C kappa and may derive from a non-V kappa or pseudo-V kappa gene fused to J kappa. Another S107 variant cDNA, however, has a proper V kappa linked in phase to J kappa (albeit missing the first two germ-line J kappa codons) and the MOPC 173 variant sequence also contains a proper V kappa--J kappa join, although it does not encode a tryptophan residue common to all immunoglobulin chains. The presence of two potentially expressible kappa mRNAs in both S107 and MOPC 173 suggests that allelic exclusion does not hold in all lymphocytes, or that it sometimes reflects events subsequent to mRNA production, such as inability of certain kappa chains to assemble properly with the heavy chain. These observations are compatible with a stochastic model for allelic exclusion in which productive and nonproductive V--J recombination events occur at a certain frequency for each allele.

Alleles↗
PubMed1993

Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5' end.

Fibrillin is an important structural protein of the extracellular matrix. It is a large cysteine-rich glycoprotein with extensive intrachain disulfide bonds, likely contributed by multiple EGF-like repeats. We have previously published 6.9 kb of FBN1 cDNA sequence. FBN1 cDNA clones that extend the sequence 3089 bp in the 5' direction are described in this report. The deduced primary structure suggests that fibrillin is composed of multiple domains. The most predominant feature is the presence of 43 calcium binding EGF-like repeats. We demonstrate here that fibrillin molecules bind calcium. In addition, three alternatively spliced exons at the 5' end are described. Analysis of 5.8 kb of surrounding genomic sequence revealed a 1.8-kb CpG island spanning the alternatively spliced exons and the next downstream exon. Since FBN1 is the gene responsible for Marfan syndrome, the information presented here will be useful in identifying new mutations and in understanding the function of fibrillin in the pathogenesis of the disease.

Alternative Splicing↗
PubMed2002

Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli.

An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL. On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library. A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned. Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E. coli), recombinant ALDH2 and ALDH2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E. coli chaperonin proteins. Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively. Co-expression of ALDH2 with ALDH2(2) in the presence of E. coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer. Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation. Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system. The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly. Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.

Aldehyde Dehydrogenase↗
PubMed1996

Identification of a novel human gene containing the tetratricopeptide repeat domain from the Down syndrome region of chromosome 21.

The Down syndrome (DS) region on chromosome 21, which is responsible for the DS main features, has been defined by analysis of DS patients with partial trisomy 21. Within the DS region, we constructed a 1.6-Mb P1 contig map previously. To isolate gene fragments from the 1.6-Mb region, we performed direct cDNA library screening and exon trapping using the P1 clones and a human fetal brain cDNA library, and obtained 67 cDNA fragments and 52 possible exons. Among them, 23 cDNA fragments and 4 exons were interpreted to be derived from a single gene by localization on P1 clones and by Northern analysis. To obtain the full-length cDNA sequence, longer cDNA clones were further screened from another human cDNA library which was enriched with longer cDNA species. These clones were sequenced and assembled to a sequence of 9045 bp. This transcribed sequence encodes a novel 2025 amino-acid protein containing tetratricopeptide repeat (TPR) motifs and therefore the gene was designated as TPRD (a gene containing the TPR motifs on the Down syndrome region). The TPR domain has been found in a certain protein phosphatase and in other proteins involved in the regulation of RNA synthesis or mitosis. The TPRD gene, the novel gene which was proved to be in the 1.6-Mb region and to have the interesting features described above, is a candidate for genes responsible for the DS phenotypes.

Amino Acid Sequence↗
PubMed1991

Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification.

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R-PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Amino Acid Sequence↗
PubMed1990

Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation.

Glucocorticoid receptors are ligand-dependent transcription factors that are subject to down-regulation by their cognate ligand; however, the mechanisms mediating this physiological response are not completely understood. Since analysis of the human glucocorticoid receptor (hGR) cDNA sequence revealed the presence of sequences with homology to both positive and negative glucocorticoid regulatory elements, we have examined the potential of hGR to bind to the hGR cDNA by Southwestern blot analysis. The data revealed that glucocorticoid receptors exhibited specific binding to their own cDNA. To determine whether this binding was of functional significance in the down-regulation of glucocorticoid receptors, we analyzed the effect of glucocorticoids on hGR protein levels from COS 1 cells transfected with an hGR cDNA expression vector. These transfected cells produced intact hGR that were capable of ligand-dependent regulation of a co-transfected glucocorticoid-responsive reporter gene. Glucocorticoid treatment of hGR-transfected cells resulted in down-regulation of hGR (assayed by both glucocorticoid binding capacity and hGR protein levels) within 24 h of steroid administration. To determine if the glucocorticoid-induced down-regulation of transfected hGR was compatible with effects at the levels of receptor gene expression and RNA stability, we examined hGR mRNA steady state levels. Reductions from 2- to 6-fold were observed in hGR mRNA levels following glucocorticoid treatment of transfected COS 1 cells. This down-regulation of transfected hGR mRNA could not be attributed to either the Rous sarcoma virus promoter, which drives hGR expression, or to other sequences present in the vector plasmid since transcription of a related plasmid containing a chloramphenicol acetyltransferase gene in place of the hGR cDNA was not regulated by glucocorticoids. Down-regulation of hGR mRNA by glucocorticoids in transfected cells occurred in a time- and dose-dependent manner that is consistent with a glucocorticoid receptor-mediated process. Glucocorticoid-induced down-regulation of hGR mRNa steady state levels was not observed in COS 1 cells transfected with cDNAs encoding mutant hGR (defective in either steroid or DNA binding), which indicates that functional steroid and DNA binding domains of the expressed hGR were required for down-regulation. Interestingly, treatment of transfected COS 1 cells with the glucocorticoid antagonist RU486 also resulted in down-regulation of transfected hGR mRNA. Deletion analysis revealed that the region of the hGR cDNA that was responsible in part for the observed down-regulation in response to glucocorticoid was contained within a 1-kilobase restriction fragment (from base pair +527 to +1526).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗