A COMBINATION OF DISTAL AND PROXIMAL REGIONS IS REQUIRED FOR EFFICIENT PROLACTIN REGULATION OF TRANSFECTED RABBIT ALPHA-S(1)-CASEIN CHLORAMPHENICOL ACETYLTRANSFERASE CONSTRUCTS

In the rabbit, alpha s(1)-casein is the major casein secreted in the m ilk. Transcription of the alpha s(1)-casein gene is induced by PRL. To define the positions of the cis-sequences involved in the control of rabbit alpha S-1-casein gene expression by PRL, chimeric genes contain ing upstream regions of alpha s(1)-casein gene linked to the chloramph enicol acetyltransferase gene were cotransfected into Chinese hamster ovary cells with the plasmid expressing the rabbit mammary PRL recepto r, It was observed that a distal fragment -3442/-3118 was responsible for a high induction of PRL sensitivity when linked in the 5'-position to a chimeric construct (-391/1774)-chloramphenicol acetyltransferase . A cooperation between distal and proximal regions of the asl-casein gene is responsible for the PRL-dependent enhancer activity of the dis tal fragment. The mammary gland-specific nuclear factor-like binding s equence found around position -90 in the proximal promoter of the alph a s(1)-casein gene is involved in this cooperation. The distal fragmen t was further studied to determine the position of regulatory regions. A -3442/-3385 fragment was sufficient to induce a PRL sensitivity sim ilar to that conferred by the larger -3442/-3118 distal fragment, but multiple interactions are likely to exist between other regulatory reg ions included in this distal fragment. Four DNA-binding regions (I-IV) have been identified within the reduced -3442/-3385 fragment by footp rint experiments using rabbit mammary gland or liver nuclear extracts (NE). Protected area III is observed using both NE. Protected areas 1, II, and IV are specific for lactating mammary gland NE. The sequences of areas I and IV share several homologies with the sequence of the m ammary gland-specific nuclear factor-binding site.