Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-alpha 1(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-alpha 1(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10(-9)-10(-7) M) or insulin (10(-9)-10(-6) M) on mRNA species for osteonectin or pro-alpha 1(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of 10(-9) M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5' flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by 10(-8) and 10(-7) M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor beta (TGF-beta, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-alpha 1(I)-collagen in a dose-dependent manner. Transforming growth factor alpha (TGF-alpha) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-alpha 1(I)-collagen mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Insulinlike growth factor 1 regulates mRNA levels of osteonectin and pro-alpha 1(I)-collagen in clonal preosteoblastic calvarial cells. 220 53

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.
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PMID:Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production. 222 25

Tissue-specific expression of the rat growth hormone (rGH) gene requires binding of a pituitary-specific factor. Binding of this factor has been used to explain tissue-specific expression of the human growth hormone (hGH-N) gene in transfected rat pituitary (GC) tumour cells. Neither rat fibroblast (R2) nor human cervical carcinoma (HeLa) cells contain the rat pituitary-specific factor. Thus, no expression of hGH-N or rGH would be expected in these cells. R2 cell lines containing stably integrated hGH-N or rGH genes were generated. Expression of hGH-N but not rGH was detected. By contrast, stably transfected HeLa cells did not express the endogenous or transfected hGH-N genes. However, an hGH-N transcript was detected when hGH-N gene expression was directed by a viral promoter. This suggests that the block in expression occurs at the level of transcription and not mRNA stability. Hybrid genes containing 496 base pairs (bp) of hGH-N or 234 bp of rGH 5'-flanking DNA, including promoter sequences, fused to the bacterial gene coding for chloramphenicol acetyltransferase were used to stably transfect R2 cells. The hybrid hGH-N gene was more active than a promoterless construction in these cells. By contrast, the hybrid rGH gene was not. These data suggest that the hGH-N gene can be activated by rat transcription factors other than those found in pituitary cells.
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PMID:Human growth hormone gene expression in rat but not human non-pituitary cells after stable gene transfer. 232 31

We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.
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PMID:Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression. 239 70

Release of prolactin from both normal pituitary cells and rat pituitary tumor (GH) cells is an osmotic process that is dependent upon chloride. The long term growth rate of GH-cells in medium in which chloride was exchanged with isethionate was completely normal, but, by 48 h, isethionate substitution resulted in a 70% decrease in the concentration of internal and secreted prolactin. Isethionate caused a much smaller reduction in growth hormone production (less than 20%). These results suggest that exchange of chloride with isethionate is inhibiting the synthesis of prolactin. Reduction of intracellular levels of prolactin in cells grown in isethionate-containing medium was evident by 30 h, and the level of prolactin was reduced 92% at 96 h. This reduction in the internal concentrations of prolactin was reversed when the cells were returned to normal medium containing chloride with a t1/2 of 48 h. Addition of epidermal growth factor and the calcium channel agonist BAY K 8644 to cells in medium containing chloride increased internal prolactin by 400%, and isethionate exchange reduced the response by 85%. To confirm that isethionate exchange was inhibiting the synthesis of prolactin, mRNA concentrations for prolactin and actin were determined. Both basal and hormone-stimulated levels of prolactin mRNA were reduced 70 to 90% by isethionate exchange, while actin mRNA levels did not change. To determine whether the effect of isethionate was at the level of gene transcription, GH-cells were transfected with a prolactin-chloramphenicol acetyltransferase fusion gene and chloramphenicol acetyltransferase expression was assessed using cells in chloride and isethionate-containing media. Both basal and hormone-stimulated synthesis of chloramphenicol acetyltransferase driven by the prolactin promoter was inhibited by isethionate exchange. These studies demonstrate that exchange of medium chloride with isethionate inhibits the synthesis of prolactin at the level of transcription.
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PMID:Prolactin synthesis in cultured pituitary cells is chloride-dependent. 246 Apr 42

An important physiological control of the glycoprotein hormone alpha-subunit is the negative feedback by thyroid hormones in the thyrotrope. A region of the rat glycoprotein hormone alpha-subunit gene that is involved in transcriptional regulation by thyroid hormone has been identified by transient transfection studies, and sequence-specific binding of the thyroid hormone receptor to a site within this region has been demonstrated. Deletion-mutation studies using plasmid expression vectors containing either 246, 170, or 80 base pairs of the 5'-flanking region of the rat alpha-subunit gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene demonstrate 3,5,3'-triiodo-L-thyronine (T3)-regulated expression in GH3 cells, a T3-responsive somatotrophic cell line. In order to investigate the possibility of thyroid hormone receptor interaction with this segment of the rat alpha-subunit gene, the binding of the thyroid hormone receptor to synthetic oligodeoxynucleotides was analyzed using an avidin-biotin complex DNA binding assay. An oligodeoxyribonucleotide representing a fragment of the alpha-subunit gene from -74 to -38, relative to the transcriptional start site, shows significant binding to [125I]T3-receptor complex present in nuclear extracts of GH3 cells. This fragment binds receptor to a degree similar to that seen with a fragment of the rat growth hormone gene which contains a putative thyroid hormone-responsive element. In addition, this fragment of the rat alpha-subunit gene binds to the in vitro synthesized human c-erbA beta protein, which has been identified as a member of the family of putative T3 receptors. These data demonstrate that a cis-active thyroid hormone-responsive element resides in the 5'-flanking region of the rat alpha-subunit gene and that the mechanism involved in the suppression of expression of this gene by T3 could involve specific binding of the thyroid hormone receptor to this region of the gene.
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PMID:Thyroid hormone regulation of the rat glycoprotein hormone alpha-subunit gene promoter activity. 246 63

In DNA cotransfection experiments, the Epstein-Barr virus immediate-early gene product, BMLF1, stimulated the chloramphenicol acetyltransferase (CAT) activity of both latent and productive EBV promoters linked to CAT. This BMLF1-induced increase in CAT activity was out of proportion to the effect on CAT mRNA, suggesting a posttranscriptional mechanism. Furthermore, when growth hormone was used as a reporter gene instead of CAT, BMLF1 no longer functioned. Thus, the BMLF1 effect was reporter-gene dependent. The effect of the BMLF1 gene product does not then appear to be directed at promoter activation, but instead may function to increase the level of an as yet unidentified protein(s) required for Epstein-Barr virus infection.
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PMID:The Epstein-Barr virus immediate-early gene product, BMLF1, acts in trans by a posttranscriptional mechanism which is reporter gene dependent. 254 2

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.
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PMID:Specific gene suppression by engineered ribozymes in monkey cells. 255 2

The regulation of gastrin gene transcription was studied in GH4 pituitary cells transfected with constructs comprised of the first exon of the human gastrin gene and various lengths of 5' regulatory sequences ligated upstream of the reporter gene chloramphenicol acetyltransferase. Gastrin reporter gene activity in GH4 cells was equal to the activity of a reporter gene transcribed from the endogenously expressed growth hormone promoter. The effect of a variety of peptides on gastrin gene transcription including epidermal growth factor (normally present in the gastric lumen), gastrin-releasing peptide, vasoactive intestinal peptide, and somatostatin (present in gastric nerves) was assessed. Epidermal growth factor increased the rate of gastrin transcription almost 3-fold, whereas thyrotropin-releasing hormone and vasoactive intestinal peptide increased gastrin transcription 2- and 1.5-fold, respectively. Gastrin-releasing peptide, a peptide that strongly stimulates gastrin release, weakly increased gastrin transcription (1.3-fold). Somatostatin inhibited the increase in gastrin transcription induced by epidermal growth factor, thyrotropin-releasing hormone, and vasoactive intestinal peptide. Constructs containing various lengths of 5' regulatory sequences defined a response element -40 to -82 base pairs (bp) 5' to the transcription initiation site. This 40-bp sequence contains Sp1 and AP2 binding sites, which suggests that epidermal growth factor and thyrotropin-releasing hormone stimulate gastrin gene transcription through transcription factors that bind to Sp1 and/or AP2 motifs.
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PMID:Regulation of the gastrin promoter by epidermal growth factor and neuropeptides. 256 64

Somatostatin is a peptide synthesized in the pancreatic islets, nervous system, gastrointestinal tract, and thyroid gland. Factors that control islet cell-specific expression of the somatostatin gene were analyzed by expression of fusion genes consisting of 5' rat somatostatin gene sequences linked to coding sequences of the receptor genes, bacterial chloramphenicol acetyltransferase, and human growth hormone. Fusion genes containing 900 and 250 base pairs (bp) of 5'-flanking DNA were preferentially expressed at 5-10-fold higher levels in somatostatin-producing islet cell lines, as compared with islet cell lines that produced insulin and glucagon, and in three non-islet cell lines. A deletional mutation consisting of only 65 bp of 5'-flanking sequence of the rat somatostatin gene expressed in all islet cell lines but not in non-islet lines, indicating the existence of a negative-acting islet cell-specific element located between nucleotides -250 and -65. The 65-bp sequence contains the octameric cAMP-responsive enhancer (CRE) TGACGTCA (nucleotides -48 to -41). Fine mapping of sequences responsible for islet-specific expression by substitution of synthetic oligonucleotide cassettes revealed full retention of expression by deletion to nucleotides -48 and complete loss of expression at nucleotides -42 of the CRE. Substitution of the 9 bp adjacent 3' to the CRE of the somatostatin gene (nucleotides -40 to -32) with the corresponding sequence located 3' to the CRE of the glucagon gene abolished expression. By gel mobility shift and DNaseI footprinting analyses, proteins in extracts of islet cells bound to the 24 bp including the CRE and downstream adjacent 9 bp (nucleotides -58 to -35). An additional upstream region of DNA was protected from DNase I digestion (nucleotides -110 to -80). Proteins from non-islet cells bound to the region from nucleotides -58 to -35, but patterns of DNase I protection differed from those using proteins from islet cells. These observations indicate that several DNA-binding proteins interact with cis-acting elements located between 35 and 58 bp upstream of the transcriptional start site of the rat somatostatin gene to determine islet cell-specific gene expression. CRE-binding protein(s) is ubiquitous among phenotypically different cells, and expression of the somatostatin gene in non-somatostatin-producing islet cells appears to be inhibited by a negative-acting element located upstream of the CRE.
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PMID:Somatostatin gene expression in pancreatic islet cells is directed by cell-specific DNA control elements and DNA-binding proteins. 256 13


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