The hamster ductus deferns smooth muscle tumor cell line (DDT1MF-2) contains androgen receptors and is stimulated by androgens to grow. In the presence of the androgen methyltrienolene, the level of androgen receptor increases to a steady state concentration of 20 fmoles/mug DNA (2.0 mg of receptor/1011 cells). The androgen receptor from these cells will be purified by a combination of preparative denaturing Slab gel electrophoresis, affinity chromatography, photoaffinity labeling, and preparative two-dimensional polyacrylamide gel electrophoresis. A phage lambda gt11 complementary DNA library with over 250,000 independent recombinant has been prepared from messenger RNA, isolated from androgen stimulated DDT1 cells. This library has been shown to contain full length inserts to other low abundance messenger RNAs. A new lambda gt11 library to androgen stimulated cells mRNA which have been pulsed 1-2 hr with 1 mug/ml cycloheximide will also be prepared. We will generate at least 2 x 10/6 independent recombinants. Cycloheximide treatment has previously been shown to stimulate estrogen receptor and glucocorticoid receptor messenger RNA. We will also prepare a lambda gt10 library to just androgen stimulated DDT1 messenger RNA. The pure androgen receptor will be used to determine amino acid composition and the amino acid sequence of various peptides. Several oligonucleotide probes (20-40 nucleotides long) will be prepared with minimal codon redundancy. These probes will be used to screen the lambda gt11- cDNA and a lambda gt10 libraries and to isolate and sequence the cDNA for the androgen receptor mRNA. The cDNA to the putative androgen receptor messenger RNA will be verified by at least 4 criteria to establish, infact, it does contain the coding information for the androgen receptor. The cDNA will then be used to isolate the genomic fragments for the androgen receptor gene(s) and identify the promoter region(s). The cDNA for this hamster androgen receptor mRNA will also be utilized to screen a lambda gt11-cDNA and lambda gt10 libraries made to human benign prostate hyperplasia messenger RNA. The cDNAs will then be used to study androgen receptor gene expression to growth and differentiation in both normal and abnormal tissues and in cells in a varieity of androgen responsive systems.
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