Biomedical subjects

S Radovick

Publications and source records attributed to S Radovick.

40 records · Page 3Linked to original sources

PubMed1989

Thyroid hormone inhibition of human thyrotropin beta-subunit gene expression is mediated by a cis-acting element located in the first exon.

Thyroid hormone regulation of the human thyrotropin beta-subunit gene (TSH beta) was examined in a human embryonal cell line (293). Transient expression studies were performed with chimeric plasmids containing the reporter gene, chloramphenicol acetyltransferase. Sequences in the first exon between +9 and +37 base pairs (bp) enhanced gene expression from the human TSH beta promoter in the absence of thyroid hormone as well as mediated a concentration-dependent triiodothyronine (L-T3) decrease in gene expression. Thyroid hormone inhibition of expression was also conferred to the herpes simplex virus thymidine kinase promoter by inserting +3 to +37 bp of the human TSH beta gene downstream from the start of transcription. Primer extension analysis of RNA from transfected cell cultures revealed accurate transcription initiation in only those constructs which contained sequences between +9 and +37 bp. Moreover, RNA analysis confirmed that L-T3 inhibition of chloramphenicol acetyltransferase activity from chimeric pTSH beta CAT constructs occurred at a pretranslational level. In addition, a nuclear thyroid hormone receptor, c-erbA-beta, bound to this region in an avidin-biotin DNA binding assay. These data suggest that L-T3, bound to its receptor, may inhibit human TSH beta expression by interfering with an element that functions to enhance gene expression.

Chloramphenicol O-Acetyltransferase↗

PubMed1989

Pre-translational and post-translational regulation of TSH synthesis in normal and neoplastic thyrotrophs.

We are interested in the mechanisms by which endocrine and developmental factors regulate TSH synthesis at both pre-translational and post-translational levels. Thyroid hormone profoundly decreases transcription of the TSH-beta gene, while TRH and agents modifying cyclic AMP increase transcription. To elucidate the molecular mechanisms underlying these effects, human embryonal kidney cells were transfected with constructs of the human TSH-beta gene fused to the chloramphenicol acetyltransferase gene. The first exon of human TSH-beta, contains an element that increases basal expression and mediates T3-induced gene repression, probably through a direct interaction with c-erbA beta. This transcriptional repression by T3 appears aberrant in thyrotropic tumors. In contrast, TRH and agents modifying cyclic AMP mediate increased transcription of TSH-beta through interacting with upstream regulatory elements. Thyroid hormone, TRH and developmental factors also regulate the branching pattern and relative sialylation of TSH carbohydrate chains, which may affect TSH action in vitro and in vivo. Certain thyrotropic tumors produce TSH with more complex carbohydrate branching patterns, which may increase its biologic activity.

Humans↗

PubMed1988

Isolation and characterization of the human thyrotropin beta-subunit gene. Differences in gene structure and promoter function from murine species.

The human thyrotropin beta-subunit gene was isolated and characterized from two genomic libraries and found to contain three exons separated by two introns of 3.9 and 0.45 kilobase pairs. Exons 2 and 3 in the mouse thyrotropin beta-subunit gene are not found in humans due to a lack of consensus sequences important in exon splicing. Moreover, using primer extension, RNA sequencing, and S1 nuclease analysis, we determined, in a thyrotropin-producing pituitary adenoma, that exon 1 in humans contains only one transcriptional start site and is 37 base pairs in length. This is unlike both the rat and mouse thyrotropin beta-subunit genes which contain two transcriptional start sites. Changes in the genomic structure of the more 5' "TATA box" and surrounding "CAAT box" might explain why the more 5' start site in humans is apparently not utilized. Moreover, the first exon in humans is longer than the corresponding exon in murine species presumably due to a 9-base pair insertion between the TATA box and transcriptional start site (37 versus 27 nucleotides). Thus, while alternative exon splicing and differential start site utilization in response to thyroid hormone may be important in the regulation of murine thyrotropin beta-subunit genes, they are not found in man.

Adenoma↗

PubMed1988

Cloning of the human thyrotropin beta-subunit gene and transient expression of biologically active human thyrotropin after gene transfection.

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.

Animals↗