ANALYSIS OF THE INDUCTIVE EFFECT OF THE GENOMIC EQUIVALENT OF HALF1 SEQUENCE IN THE REVERSION OF RAT DEDIFFERENTIATED HEPATOMA-CELLS

HALF1 (human activator of liver function 1) is a closed DNA duplex imp licated in reversion of rat dedifferentiated C2 hepatoma cells to a we ll-differentiated state. A copy of HALF1 is found in high-molecular-we ight DNA in the human genome. The genomic equivalent of HALF1 and its flanking sequences [gH(5'-3') fragment] have previously been cloned an d sequenced. To analyze the ability of the gH(5'-3') fragment to induc e reversion process of C2 cells, two series of transfections were perf ormed: (1) cotransfection of gH(5'-3') and plasmid pSVneo1 and (2) tra nsfection of gH(5'-3') inserted into pSVneo1. The frequency of reversi on was enhanced in transfected cells from the first experiment whereas no revertants were obtained from transfected cells in the second one. DNA analysis of the revertant clones revealed that reversion is assoc iated with the transient presence of nonintegrated gH(5'-3') molecules . C2 cells were also transfected with gH(5'-3') cloned in pSV2dhfr. In the products of this transfection, the genesis of revertants correlat ed with amplification process of the gH(5'-3') sequence. We conclude t hat the presence of integrated copies of gH(5'-3'), even in high copy number, is not sufficient to induce the reversion process. We propose that extrachromosomal forms of gH(5'-3'), either given to cells or for med during amplification cycles, are involved in the reversion process of C2 cells. (C) 1994 Academic Press, Inc.