ABSTRACT

A number of structurally very similar pheromone-binding proteins (major urinary proteins; MUPs) are synthesized in mouse liver and rapidly excreted in the urine. Male and female inbred mice display different characteristic patterns of MUP expression. Here we present a detailed study of the RNA and protein products corresponding to specific MUP genes previously isolated from genomic DNA of the Balb/c strain. By in vitro transcription of equivalent cDNA clones, translation of the resulting RNA in the reticulocyte lysate system and isoelectric focusing, the protein products of genes BL1, BS1 and BS6 were shown to be MUP 2a, MUP 2b and MUP 4 respectively. MUPs 2a and 2b were shown to be abundant both in Balb/c male urine and among the translation products of total Balb/c male liver mRNA. Two oligodeoxynucleotide probes, oBL1A and oBS1, selective for BL1 and BS1 mRNA respectively, were chemically synthesized. mRNA that hybridized with these probes (oBL1A mRNA and oBS1 mRNA) was present at different characteristic levels in the Balb/c and C57BL/6 inbred strains. In both strains the level of expression was much higher in males than females and the male/female expression ratio of oBS1 RNA was higher than that of oBL1A RNA. Comparison of these mRNA levels with the amounts of different MUP proteins present in urine and the translation products of liver mRNA indicated that proteins other than MUP 2a and MUP 2b are coded for by the C57BL/6 oBL1A and oBS1 mRNAs.

C57BL/6 mice homozygous for the lit mutation are GH deficient and transcribe MUP genes at a level much lower than that obtaining in normal mice of either sex, indicating that transcription is induced by GH in both males and females. When lit/lit mice were treated with GH under two different regimes, MUP gene transcription was partially induced to different degrees and the level of oBL1A mRNA was induced more highly than that of oBS1 mRNA. Thus there exists a correlation between the inducibility of these mRNAs and their level of expression in females relative to males; oBL1A mRNA is both more highly expressed in females and more readily induced by GH than oBS1 mRNA. This suggests that the male and female expression patterns are due to differential inducibility of different MUP genes together with a stronger inducing stimulus in males. GH administered continuously by infusion repressed MUP gene expression. We interpret this to mean that induction is due to intermittent GH stimulation in both sexes and that the longer interpulse interval reported to occur in males leads to more effective induction than the shorter interpulse interval observed in females.

ABSTRACT

Three regions required for the expression of a mouse major urinary protein (MUP) transgene were identified by a deletion analysis. One of these was located upstream of the cap site between −2139 and −1800, another was the proximal promoter region downstream of −324 and the third lay within the 338 nucleotide intron 1. Both the proximal promoter and intron 1 are involved in sexually dimorphic expression of the transgene (male/female ratio 20), which is dictated by the different temporal profiles of circulating GH in the two sexes. The data also indicated that the region between exons 3 and 7 may contribute to full expression in males and that a region between −718 and −324 may contribute towards the low expression level that obtains in females, but compared with the three principal regions the effects of these regions are relatively minor. We propose (1) that full expression of the transgene requires the co-operation of transcription factors binding to the three principal regions and (2) that the difference in expression between the sexes relates to interactions between transcription factors bound to the proximal promoter and to sites in intron 1. Our results complement earlier in vitro footprinting and gel-retardation studies of the homologous rat α_2u-globulin genes. These identified a number of response elements, including putative C/EBP and AP1 sites in the proximal promoter and intron 1 respectively and three putative ΨNF-1 sites, two in the proximal promoter and one in intron 1, but proof of the functionality of these sites in regulating transcription was lacking. The proximal promoter also contained a 34 nucleotide sequence that has 70% identity with the SPI GH response element.