Experimental Cell Research 180 (1989) 220-233 Positive and Negative Regulation of a Transfected Chimeric Tyrosine Aminotransferase Gene: Effect of Copy Number THIERRY GRANGE,” JEANNE ROUX, MICHELINE FROMONT- RACINE, and RAYMOND PICTET Institut Jacques Monod du CNRS, Unit6 INSERM 257, UniuersitL Paris VII, Tour 43, 2 Place Jussieu, 75251 Paris Cedex OS, France To define a selective system for the study of rat tyrosine aminotransferase (TAT; EC 2.6.1.5) gene expression, we have introduced into cultured cells the selectable bacterial gene gpt linked to TAT gene flanking sequences. After integration in host cell DNA, the chimeric gene exhibits the same pattern of regulation as the TAT gene. In hepatoma cells, its expression is induced after glucocorticoid hormone treatment and repressed after fusion with fibroblasts. In tibroblasts, the chimeric gene is not expressed. The correct pattern of regulation is lost when the number of integrated copies is high. At copy number above 10, the transfected gene responds poorly to glucocorticoids in hepatoma cells. At copy number above 50, the gene is expressed in fibroblasts. Another gene present in the same construc- tion and controlled by the SV40 early promoter and enhancer is positively regulated by glucocorticoids in hepatoma cells but not after fusion with fibroblasts. These data indicate that in hybrid cells, both TAT promoter and glucocorticoid-responsive elements are negatively regulated. 0 1989 Academic Press, Inc. Tissue-specific gene expression is controlled mainly at the transcriptional level. To achieve that control, both positive and negative regulations can be involved. Somatic cell genetics has provided evidence for the existence of negative regula- tions: hybrid cells obtained by fusing two different cell types generally do not express the tissue-specific functions of either parent [l]. Extinction of at least some tissue-specific genes is mediated by discrete genetic loci that map on a single chromosome [2 and references therein]. The first locus so defined is involved in tyrosine aminotransferase (TAT) extinction [3]. The TAT gene is expressed specifically in liver cells [4], where its transcription is increased by glucocorticoid hormone treatment [.5]. The TAT extinguisher locus in mouse has been located on chromosome 11. It is active in fibroblasts and in hepatoma-fi- broblast hybrid cells. It affects the TAT mRNA level in tram since the mouse TAT gene is located on chromosome 8 [3, 61. The molecular basis of this extinction phenomenon has not yet been elucidated. Gene transfer experiments should help the progress toward a better under- standing of this phenomenon. In the present paper we show that the extinguisher is active on a stably transfected chimeric TAT gene, and we analyze some of the modalities of the activity of the TAT gene glucocorticoid-responsive elements (GREs). We have substituted most of the transcribed sequences in the rat TAT ’ To whom reprint requests should be addressed. Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827189 $03.00 220