Identification by cell fusion of gene sequences that interact with positive trans-acting factors.
Cell fusion experiments have implicated positive or negative regulatory factors in the cell type--specific expression of specialized endogenous genes. The inability to readily manipulate such genes has prevented characterization of the cis-acting DNA sequences that interact with these factors. A transfection-fusion technique, which combined stable gene transfer and formation of transient heterokaryons, was used to study this class of factors and their DNA binding sites. Messenger RNA directed by a quiescent, rat prolactin promoter region stably transferred into mouse fibroblasts was detected only after fusion to rat pituitary cells, implying that pituitary cells contain a positive cell type--specific factor or factors. Nuclear run-on assays showed that fusion activation is transcriptional. Fusion did not activate either a stably transferred rat growth hormone gene promoter or expression of the endogenous silent fibroblast prolactin or growth hormone genes. Analysis by 5'-deletion mutation identified a 30-base pair DNA sequence required for cell fusion activation of the rat prolactin promoter region. Comparison with previous results from direct cellular transfer of this region implies that transfection-fusion identifies novel regulatory DNA sequences.
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D002459:Cell Fusion; D015500:Chloramphenicol O-Acetyltransferase; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics; D010902:Pituitary Gland / Q000502:physiology*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D014157:Transcription Factors; D011388:Prolactin; D013006:Growth Hormone; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase