Abstract
The 5'-end of the human transforming growth factor-beta 1 gene (TGF-beta 1) was isolated from a human leukocyte genomic DNA library. Analysis of the transcriptional start sites of human TGF-beta 1 mRNAs by S1 mapping and primer extension revealed two major start sites 271 nucleotides from one another; several minor sites were also identified. DNA sequence analysis showed that the promoter region contains neither a "TATA" box nor a "CAAT" box, is very G+C rich, and contains 11 CCGCCC repeats. Seven putative binding sites for the transcription factor Sp1 were also identified. To determine the location of sites that may be important for the function of the TGF-beta 1 promoter, we joined the 5'-end of the TGF-beta 1 gene to the coding region for chloramphenicol acetyltransferase. The chimeric gene produced high levels of chloramphenicol acetyltransferase activity in transfected HT-1080, AKR-2B, and A-549 cells. Sequences responsible for both promotion and inhibition of transcription were located in the region extending from 1400 to 300 base pairs upstream of the first major TGF-beta 1 transcriptional start site. The 130-base pair fragment located between 453 and 323 base pairs upstream of this start site contains positive regulatory activity in all cells tested. A second promoter activity was identified in the region between the two major transcriptional start sites. These findings revealed a complex pattern of regulation of human TGF-beta 1 gene expression.
Highlights
The 5’-end of the human transforming growth fac- deduced amino acid sequences of human, murine, porcine, tor-81 gene (TGF-81) was isolated from a human leu- bovine, and simian TGF-/31mRNAs have been reported
Studies showing located in theregion extending from 1400 3to00 base tissue-specific, time-dependent localization of TGF-Pl in the pairs upstream of the first major TGF-81 transcrip- developing mouse embryo [7] suggest that expression of the tional start site
The in lane a and the 162-bp fragment in lane b represent the primer- data areexpressed as a percentage of the mean activity obtainewdith extended products corresponding to the major start sites at +1 and phTG5 (-453 to +ll),the plasmidwith thestrongestpromoter
Summary
Different sources [10, 11].The nucleotide sequences and the Cells and Culture Conditions-Human fibrosarcoma (HT-10801, mouse NIH-3T3, mink lung carcinoma (CCL-64),and hamster fibro-. Total RNA (60 pg) was coprecipitated in ethanol with 1 X lo cpm of an end-labeled DNA probe representing the HincII-TaqI human genomic fragment (-453 to +727, see Fig. 1).Hybridization was performed by dissolving the ing from the nucleotide -453 to thenucleotide +727 (see Fig. 1).This fragment was end-labeled at the5' terminus of the TaqI site prior to digestion with Him and subsequently isolated from the agarose gels. Using this probe, we were able to observe five or more S1 nuclease-resistant fragments (Fig. precipitate in 40 pl of hybridization buffer (80%formamide, 20 mM 2 A ) after hybridization with HT-1080 mRNA. The role of these multiple sites in the regulation of on 6% sequencing gels
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