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Rat prostatic binding protein genes C1, C2, and C3 were mapped on rat chromosome 5 by in situ hybridization on rat peripheral blood chromosome preparations using three different cDNA probes. Of the grains detected, 15.9%, 25.2%, and 19.6%, respectively, mapped to chromosome 5. For each probe, the label was predominantly located on 5q31, but for C2 and C3 an additional site on 5q21 was found. The results suggest that three genes coding for the different polypeptide chains of rat prostatic binding protein map to the same chromosome and presumably to the same chromosome band.

The complete sequence (2879 bp) of the androgen-controlled rat prostatic binding protein C2 gene and 1023 bp of the 5'- and 2127 bp of the 3'-flanking regions have been determined. The gene contains three exons (93, 203 and 147 bp) and two introns (1630 and 806 bp). It is flanked by two homopurine-homopyrimidine stretches of 55 and 131 nucleotides respectively, located at positions -405 and 4151. These sequences are remarkably sensitive towards S1-nuclease, indicating an altered DNA conformation under superhelical stress. Several palindromes and dyad structures are observed in the 5'-upstream region of the gene and at position -457, and 80% homology to the consensus sequence of a glucocorticoid receptor binding site is found.

The complete gene encoding the polypeptide Cl of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5\ m=' \ part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position \m=-\150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, Cl and C2A, the only obvious conserved structural element is the homopurine stretch located at position \m=-\400, although sequence motifs resembling steroid hormone response elements are present at several locations.

We have isolated and characterized seven members of the az,-globulin gene family from a rat genomic library. The 5' upstream region (up to 1250 base pairs starting from the EcoRI site in exon 2) of three clones was sequenced. The major transcriptional start points were located 25 base pairs downstream from the 'TATA' box. A very high degree of homology was observed over the entire studied region. Two of the examined genes displayed structural features which suggest that their expression may be impeded. A high degree of homology was observed between the promotor regions of a2,-globulin and those of the major urinary protein (MUP) multigene family of the mouse. A remarkable feature is the variable length of an A-rich region between the putative 'CAAT' and the 'TATA' consensus sequences. The size of this region differs markedly between MUP and a2,-globulin and between different members of the a2,-globulin gene family. Comparison of the a2,-globulin promotor with the corresponding region of other androgen-dependent genes (the C1, Cz and C3 subunits of prostatic steroid binding protein) reveals the presence of an A-rich region of homology located approximately 378 base pairs upstream from the cap site in the a,,-globulin genes. This region compares well with a sequence of putative enhancer function previously demonstrated in the a-fetoprotein promotor and in the immunoglobulin heavy chain promoter. a2,-Globulin is a protein of unknown function abundant in the urine of adult male rats but barely detectable in that of females. The majority of this protein is synthesized in the liver, secreted into the blood and, because of its small size (Mr = 18 700), excreted into the urine [l -31. The hepatic production of az,-globulin is under multihormonal control. Androgens are the most important physiological regulators but glucocorticoids, insulin, thyroxine and growth hormone are also required for normal synthesis [4-71. Oestrogens on the contrary [8, 91 and a factor secreted by ectopically transplanted pituitary glands [lo] suppress ~x~,-globulin. All these hormones control a2,-globulin synthesis mainly by regulating the hepatic level of a2,-globulin mRNA [ll-141. a2,-

The mRNA for component C1 of rat prostatic binding protein has been cloned and characterized. A partially purified mRNA fraction for this complex protein was reverse‐transcribed into double‐stranded cDNA and cloned into the PstI site of plasmid pBR 322. The 426‐base‐pair insert of the recombinant plasmid pC1A75 was completely sequenced. The coding region corresponds precisely to the 88 amino acid residues of C1 and in addition contains the information of a signal peptide of 23 residues. The 5′non‐coding region counts only 19 nucleotides and is incomplete but the 3′‐terminal non‐coding part of 60 nucleotides extends into the poly(A) tail. Sequence analysis of other C1‐positive clones indicates the presence of sequence rearrangements which must have occurred during the cloning procedure. Possible mechanisms for the generation of these cloning artefacts are discussed.

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