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PubMed1998

Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

Adult↗
PubMed1999

Investigation of the extracellular accessibility of the connecting loop between membrane domains I and II of the bradykinin B2 receptor.

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning alpha-helices. To analyze the topology of the bradykinin B2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. We found that a segment with predicted intracellular orientation according to the rhodopsin model, the connecting loop between membrane domains I and II of the bradykinin B2 receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B2 receptors. Extracellular orientation of this loop was further confirmed by the substituted cysteine accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modification by a membrane impermeant maleimide. In addition, this segment seemed to be involved in B2 receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B2 receptor activation, and (ii) site-directed antibodies to the connecting loop between membrane domains I and II were agonists. The agonistic activity of the antibodies was suppressed by the B2 antagonist HOE140 confirming the B2 specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B2 receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.

Antibodies↗
PubMed1986

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

Animals↗
PubMed1990

Tumor necrosis factor alpha activates soluble guanylate cyclase in bovine glomerular mesangial cells via an L-arginine-dependent mechanism.

Endothelium-derived nitric oxide (NO) causes vasodilatation by activating soluble guanylate cyclase, and glomerular mesangial cells respond to NO with elevations of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). We explored whether mesangial cells can be stimulated to produce NO and whether NO modulates mesangial cell function in an autocrine or paracrine fashion. Tumor necrosis factor alpha (TNF-alpha) raised mesangial cell cGMP levels in a time- and concentration-dependent manner (threshold dose 1 ng/ml, IC50 13.8 ng/ml, maximal response 100 ng/ml). TNF-alpha-induced increases in mesangial cGMP content were evident at 8 h and maximal at 18-24 h. The TNF-alpha-induced stimulation of mesangial cell cGMP production was abrogated by actinomycin D or cycloheximide suggesting dependence on new RNA or protein synthesis. Hemoglobin and methylene blue, both known to inhibit NO action, dramatically reduced TNF-alpha-induced mesangial cell cGMP production. Superoxide dismutase, known to potentiate NO action, augmented the TNF-alpha-induced effect. Ng-monomethyl-L-arginine (L-NMMA) decreased cGMP levels in TNF-alpha-treated, but not vehicle-treated mesangial cells in a concentration-dependent manner (IC50 53 microM). L-arginine had no effect on cGMP levels in control or TNF-alpha-treated mesangial cells but reversed L-NMMA-induced inhibition. Interleukin 1 beta and lipopolysaccharide (LPS), but not interferon gamma, also increased mesangial cell cGMP content. Transforming growth factor beta 1 blunted the mesangial cell response to TNF-alpha. TNF-alpha-induced L-arginine-dependent increases in cGMP were also evident in bovine renal artery vascular smooth muscle cells, COS-1 cells, and 1502 human fibroblasts. These findings suggest that TNF-alpha induces expression in mesangial cell of an enzyme(s) involved in the formation of L-arginine-derived NO. Moreover, the data indicate that NO acts in an autocrine and paracrine fashion to activate mesangial cell soluble guanylate cyclase. Cytokine-induced formation of NO in mesangial and vascular smooth muscle cells may be implicated in the pathogenesis of septic shock.

Animals↗
PubMed1988

lambda PMV: a bacteriophage vector allowing single-step retrieval of cDNAs following expression in mammalian cells.

A general strategy has been developed for expression and rescue of cDNAs in mammalian cells. cDNA libraries are constructed in a new phage vector, lambda PMV, which contains simian virus 40 (SV40) early region promoter sequences for transcription of cDNA inserts, as well as a dominant-acting selectable marker neo. Efficient transfer of the cDNA library to mammalian cells can be achieved by phage particle-mediated transfection. Following selection in the antibiotic G418, cells expressing the phenotype of interest are identified and isolated. Rapid recovery of the transfected cDNA is achieved through cell fusion of the transduced cells with COS cells. Replication at the SV40 origin promotes excision of the integrated cDNA as small circular DNA, which after isolation in this form is used to transform bacteria to ampicillin resistance. To test this strategy, a cDNA encoding the human lymphocyte differentiation antigen CD8 was inserted into lambda PMV. CD8 expression on the surface of mouse L cells and the efficient recovery of full-length CD8 cDNA inserts confirm the feasibility of this system. It is anticipated that the single-step screening of libraries constructed in lambda PMV will allow for the isolation of rare cDNAs and will prove less laborious than methods currently available.

Bacteriophages↗
PubMed1997

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.

Amino Acid Sequence↗
PubMed1988

Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.

Animals↗
PubMed2001

Microsomal p450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure.

The amino-terminal region of microsomal P450s contains three distinct sequence motifs, the signal-anchor sequence (SA), the basic sequence (BS), and the proline-rich sequence (PR). Studies with two P450s of the CYP2C subfamily, P4502C11 (CYP2C11) and P4502C2 (CYP2C2), have indicated that upon expression in eukaryotic cells (yeast, COS cells, and insect cells), specific proline residues in PR are important for proper folding. In the present study, we have established that the PR region in a very different CYP gene family, P450c17 (CYP17), is also important for efficient folding. These studies have been carried out using expression in Escherichia coli. Using P4502C11, we have established that the folding requirements for P450s in bacteria are very similar to those in eukaryotic cells. Interestingly, when the PR from P450c17 is swapped for that of P4502C11 and visa versa, complete misfolding is observed. However, both the BS and SA can be swapped between these P450s without affecting folding. After proper folding of P450c17, removal of the PR by factor Xa protease has no effect on the maintenance of the P450 structure. Inspection of the sequences of many different CYP gene families indicates that the PR sequence is conserved within a gene family but varies considerably between families. We conclude that PR is important for directing the folding pathway leading to the functional P450, but not for maintaining the functional form.

Cytochrome P-450 Enzyme System↗
PubMed1985

Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins.

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.

Amino Acid Sequence↗
PubMed2006

Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

Alkaline Phosphatase↗
PubMed1983

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus.

Transcriptional regulatory elements within the Rous sarcoma virus long terminal repeat were examined by the construction of a series of deletions and small insertions within the U3 region of the long terminal repeat. The analysis of these mutations in chicken embryo cells and COS cells permitted the identification of important transcriptional regulatory elements. Sequences within the region 31 to 18 base pairs upstream of the RNA cap site (-31 to -18), encompassing a TATA box-like sequence, function in the selection of the correct site of transcription initiation and, in addition, augment the efficiency of transcription. These sequences are essential for virus replication. Sequences within the region -79 to -59, overlapping a CAAT box-like sequence, are not required for virus replication and have no obvious effect on viral RNA transcription in the presence of an intact TATA box. However, in mutants lacking a functional TATA sequence, mutations in this region serve to decrease the efficiency of correct transcriptional initiation events.

Animals↗
PubMed1993

Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase.

Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1992

An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion.

The T4-binding globulin-Gary (TBG-G) variant has severely impaired T4 binding, is unstable at 37 C, and presents an apparent anodal shift of all isoforms when submitted to isoelectric focusing. Inheritance of this abnormal TBG produces a profound decrease in the serum levels of native TBG with reciprocal changes in its denatured form, causing thyroid hormone concentrations to be as low as those found in complete TBG deficiency. The TBG-G gene possesses a single nucleotide substitution replacing the normal IIe96 (ATC) with Asn (AAC), thus creating a new site for N-linked glycosylation. In order to determine whether TBG-G contains an additional carbohydrate chain as indirectly suggested by the isoelectric focusing results, cDNAs containing the normal TBG (TBG-N), and TBG-G were inserted in the appropriate vectors to allow their expression in mammalian cells (COS-1) and in amphibian (Xenopus) oocytes. In both systems, expression of TBG-G yielded a larger molecule than TBG-N when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. However, both were identical in size when synthesized in COS-1 cells in the presence of tunicamycin or when deglycosylated after their synthesis in Xenopus oocytes. Pulse chase experiments revealed impaired secretion and excessive overall intracellular degradation of TBG-G relative to TBG-N. As expected from studies on serum from affected subjects, in vitro expressed TBG-G had a 10-fold lower affinity for T4. These studies prove that the new site for potential glycosylation created by the point mutation in TBG-G is indeed glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Mitochondria are morphologically heterogeneous within cells.

Mitochondria play key roles in the life and death of cells. We investigated whether mitochondria represent morphologically continuous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or tetra-methyl rhodamine ethyl ester (TMRE). The mitochondria of pancreatic acinar, porcine aortic endothelial (PAE) cells, COS-7 cells and SH-SY5Y cells and neocortical astrocytes all displayed heterogeneous distributions and were of varying sizes. In general, there was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could clearly be seen. DsRed1 was found to be highly mobile within the matrix of individual mitochondria, with an estimated linear diffusion rate of 1 micro m s(-1). High-intensity irradiation of subcellular regions bleached the fluorescence of mitochondrially targeted DsRed1, but did not cause the mitochondria to depolarise or fragment. A lack of rapid fluorescence-recovery-after-photobleaching (FRAP) of DsRed1 indicated lumenal discontinuity between mitochondria. We observed a slow (half-time approx. 20 min) recovery of DsRed1 fluorescence within the irradiated area that was attributed to mitochondrial movement or fusion of unbleached and bleached organelles. Mitochondria were not electrically coupled, since typically only individual mitochondria were observed to depolarise following irradiation of TMRE-loaded cells. Our data indicate that the mitochondria within individual cells are morphologically heterogeneous and unconnected, thus allowing them to have distinct functional properties.

Animals↗
PubMed1995

[Flow cytometry and laser scanning confocal microscopy analysis of the receptor distribution].

To study the physiological regulation of the receptor protein, a fluorescent probe and detection system for alpha 1B-adrenergic receptors have been developed. By using the anti-peptide antibody developed against the alpha 1B-adrenergic receptor NH2-terminus, we have examined the agonist-regulated alpha 1B-adrenergic receptor redistribution in desensitized cells. Flow cytometry analysis showed that anti-peptide antibody against alpha 1B-adrenergic receptor specifically identifies the receptor in CHO cells, COS-7 cells that were transfected with alpha 1B-adrenergic receptor cDNA and rat hepatocytes. Using a fluoro-labeled receptor ligand, BODIPY FL-prazosin, as a probe, cell surface alpha 1-adrenergic receptor subtypes can be detected by flow cytometry. Laser scanning confocal microscopy visualized the agonist-regulated redistribution process of alpha 1B-adrenergic receptor in living cells; thus, following phenylephrine (10(-6) M) stimulation, receptor antigen at the cell surface rapidly internalized and clustered together in a cell within 30 min. The results showed that the antibody and fluoro-labeled ligand are valuable tools for studying the localization and functional role of the alpha 1-adrenergic receptor subtype.

Animals↗
PubMed2001

The distinct roles of T cell-derived cytokines and a novel follicular dendritic cell-signaling molecule 8D6 in germinal center-B cell differentiation.

Germinal center-B (GC-B) cells differentiate into memory B cells and plasma cells (PC) through interaction with T cells and follicular dendritic cells (FDC). Activated T cell and FDC play distinct roles in this process. The detailed kinetic experiments revealed that cytokines secreted by activated T cells determined the pathway of GC-B cell differentiation. IL-4 directs GC-B cells to differentiate into memory B cells, whereas IL-10 steers them into PC. FDC/HK cells do not direct either pathway, but provide signals for proliferation of GC-B cells. A novel FDC-signaling molecule 8D6 (FDC-SM-8D6) produced by FDC augments PC generation in the GC. FDC-SM-8D6-specific mAb blocked PC generation and IgG secretion but not memory B cell proliferation. COS cells expressing FDC-SM-8D6 enhanced GC-B cell proliferation and Ab secretion, which was blocked by mAb 8D6. In the cultures with B cell subsets, PC generation was inhibited by mAb 8D6 in the cultures with CD27(+) B cells but not in the culture with CD27(-) B cells, suggesting that CD27(+) PC precursor is the specific target of FDC-SM-8D6 stimulation.

Antibodies, Blocking↗
PubMed2002

[Molecular cloning and analysis of bovine prolactin full-long genomic as well as cDNA sequences].

In this report, a full-length sequence of bovine prolactin (bPRL) genomic DNA with 9388 bp, which has been accepted by GenBank (Accession Number: AF426315), was firstly cloned by Long PCR procedures. This sequence consists of 5 exons, 4 introns, 854 bp of 5' upstream regulatory region and 69 bp of 3' UTR. Accession number of protein encoded by AF426315 sequence in GenBank is AAL28075 that is composed of 229 amino acid residues, in which signal sequence resides in 1-30 sites and mature polypeptide consists of 199 amino acid residues. The recombinant plasmid containing bPRL genomic DNA was then transfected into eukaryotic cells (COS-7), followed by RT-PCR procedure. An 804 bp of bPRL cDNA containing all the encoding region was obtained, indicating that the bPRL genomic DNA reported herein had its biological function at the transcriptional level. Results derived from information searching by Blast program revealed that there were various SNP sites in the sequences of bPRL mRNAs and ESTs collected in GenBank, which located mainly in downstream encoding region and 3' UTR. These SNP sites did not alter the related amino acids encoded. In addition, mRNA sequences encoding 5' signal sequence of bPRL gene was highly conserve.

Animals↗
PubMed1989

Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor.

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.

Animals↗