The major urinary proteins (MUPs) in mice are coded for by a gene family which consists of ca. 30 members. The number of MUP genes that are expressed is not known. Previous studies have shown that MUP mRNAs are present in several secretory tissues in addition to the liver, in which they were originally identified. In this paper we show, through restriction analysis of MUP cDNAs, that distinct sets of MUP mRNAs are synthesized in each of the tissues studied and that these mRNAs are most likely coded for by different genes. As is shown, MUP mRNAs of different tissues are related to an extent that precludes the use of gene-specific probes in differentiating among them. The regions of homology also include the 3' untranslated regions of MUP mRNAs. The question of differential expression was thus investigated by searching for restriction polymorphisms in MUP mRNAs. We demonstrate that subtle differences in the sequences of even scarce mRNAs can be recognized by this particular approach. In addition, it is shown that MUP mRNAs of different tissues code for different, nonoverlapping sets of polypeptides, as determined by gel electrophoresis of in vitro-translated precursors to MUPs. The relevance of these results to models of evolution of tissue-specific regulation in a multigene family is discussed.It appears that the synthesis of tissue-specific mRNAs at the appropriate stage of development is controlled through transcriptional regulation (8, 9). The mechanisms of this regulation are not known, nor is it known how this type of regulation arose during evolution. As has been suggested (see, e.g., reference 12), detailed studies of the structure of tissue-specific genes which belong to multigene families should provide clues concerning both the evolution and the molecular basis of tissue-specific regulation. In this paper we show that the genes coding for major urinary proteins (MUPs) form such a gene family.MUPs were originally recognized because of their high concentration in the urine of adult male mice. The urinary MUPs are synthesized in the liver (13,23) under the influence of a number of hormones, including thyroxine, growth hormone, and testosterone (16,21 The MUPs are coded for by ca. 30 genes that are clustered in the Mup-a locus of chromosome 4 (1, 5). Because of the presence of a large number of MUP genes, the possibility exists that MUP genes are differentially expressed in different tissues. This question was thus investigated with the focus on expression of MUP genes in the liver and the submaxillary, sublingual, and lachrymal glands. MATERIALS AND METHODSAnimals. An inbred strain of Swiss white mice (NCS) raised at The Rockefeller University was used in all experiments.Preparation of poly(A+) RNA. Polyadenylated [poly(A+)] RNA was extracted from fresh mouse tissues by the technique of Chirgwin et al. (4) as previously described (9).RNA electrophoresis and blotting. The procedures used were those of D. Goldberg and B. Seed (unpublished) and Thomas (25). Poly(A+) RNA was denatured by heating for 5 min at ...