Nucleotide sequences of liver, lachrymal, and submaxillary gland mouse major urinary protein mRNAs: mosaic structure and construction of panels of gene-specific synthetic oligonucleotide probes.

The mouse major urinary proteins (MUPs) are encoded by a gene family of about 35 to 40 members. MUPs are synthesized in at least six secretory tissues under a variety of developmental and endocrine controls, but the identities of the individual genes expressed in each tissue have not previously been established. In this article, we present the nucleotide sequences of five MUP mRNAs which we designate MUP I through V. MUPs I, II, and III are the most abundant MUP mRNA species in the liver, and MUPs IV and V are the most abundant MUP mRNA species in the lachrymal gland and the submaxillary gland, respectively. The sequence data show that each of the five mRNAs is encoded by a distinct member of the gene family. The structures of the MUP mRNA consist of interspersed segments of variable and conserved sequences. On the basis of the sequences of the variable segments, gene-specific panels of synthetic oligonucleotide probes were prepared. The gene-specific panels were used to identify cloned genes and, as described in the accompanying paper (K. Shahan, M. Denaro, M. Gilmartin, Y. Shi, and E. Derman, Mol. Cell. Biol. 7:1947-1954, 1987), to characterize the expression of MUP genes I through V.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.     

Tissue-specific expression of major urinary protein (MUP) genes in mice: characterization of MUP mRNAs by restriction mapping of cDNA and by in vitro translation.

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.     

T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of serum protein gene expression in mouse visceral yolk sac and foetal liver.

We have shown by molecular hybridisation that the mRNAs for albumin, transferrin, apolipoprotein-A1, and alpha 1-antitrypsin are expressed at high levels in mouse visceral yolk sac. In contrast, the mRNAs for contrapsin (a plasma protease inhibitor) and the major urinary proteins (MUPs) are not detected in the visceral yolk sac at any stage of embryonic development. Contrapsin and MUP mRNAs both appear late in liver development. These differences in expression suggest that the visceral yolk sac is more similar to the foetal than adult mouse liver in its pattern of gene expression. However, the developmental time course of expression of these mRNAs is different between the foetal liver and the yolk sac. Evidence is also presented that the visceral yolk sac synthesises and secretes other apolipoproteins in addition to apolipoprotein-A1. These results suggest that the visceral yolk sac and foetal liver, two tissues with different embryological lineages, perform similar functions but are independently programmed for expression of the same set of serum protein genes.     

Major urinary proteins, alpha(2U)-globulins and aphrodisin

The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as alpha(2U)-globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded beta-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 10(4)-10(5) M(-1) range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics.     

Multiple genes coding for the androgen-regulated major urinary proteins of the mouse.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.     

Biosynthesis of the major urinary proteins in mouse liver: A biochemical genetic study

By labeling liver protein in vivo with [³H]leucine, the relative biosynthetic rate has been measured for the major urinary proteins (MUPs), three closely related, androgen-regulated proteins that are synthesized in mouse liver, secreted into the bloodstream, and excreted into the urine. In livers from females of strain C57BL/6J, total MUP synthesis represents about 0.6–0.9% of the total protein synthesis; in males and testosterone-treated females of the same strain, synthesis increases to about 3.5–4.0% of the total. This 4-to 6-fold induction of total MUP synthesis is similar to the androgen-mediated increase in MUP-specific messenger RNA reported by others, and indicates that the previously observed 20- to 25-fold induction of total MUP excretion into urine is generated partly at the posttranslational level. By measuring the ratio of synthesis of the individual MUPs, it was determined that the testosterone-mediated change in the relative levels of the MUPs in urine reflects a similar change in the pattern of MUP synthesis, indicating that the posttranslational processes operate on the quantity, and not the nature, of MUPs excreted. A survey of seven inbred mouse strains revealed polymorphism for the rate of total MUP synthesis in untreated females. Two classes could be distinguished on the basis of a 3- to 5-fold difference in the rate. This variation does not correlate with variation at Mup-a, a locus that controls the ratio of the three MUPs in urine from androgen-induced mice. These findings are consistent with the notion that MUP expression is controlled by a variety of independently assorting genes.     

Rat α2u-globulin mRNA expression in the preputial gland

Rat _2u-globulin and the mouse major urinary proteins (MUP) are encoded by homologous multigene families whose members exhibit diverse tissue-specific, developmental, and hormonal controls of expression. Although their patterns of expression and hormonal control appear to be very similar in many respects, we have found high levels of _2u-globulin mRNA in rat preputial glands, whereas MUP mRNA could not be detected in the male mouse preputial gland. Male and female rat preputial have similar concentrations of _2u-globulin mRNA, suggesting an absence of endocrine regulation as occurs in the liver and lachrymal glands. Two-dimensional polyacrylamide gel electrophoresis of proteins encoded by hybrid-selected _2u-globulin mRNA indicates that the liver and lachrymal translation products have different mobilities. However, many of the preputial gland products comigrate with most or all of the liver and lachrymal products. Among the possibilities suggested by these results is that _2u-globulin genes expressed in liver and lachrymal glands under endocrine control are also expressed constitutively in the preputial gland.This work was supported by Public Health Service Research Grant GM25023 from the National Institutes of Health.     

Identification and characterization of functional genes encoding the mouse major urinary proteins.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.     

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.     

Subfamily of submaxillary gland-specific Mup genes: chromosomal linkage and sequence comparison with liver-specific Mup genes

Mouse major urinary proteins (MUPs) are encoded by a family of ca. 35 genes that are expressed in a tissue-specific manner in several secretory organs; in the liver, in the submaxillary, sublingual, parotid and lachrymal glands, and in the skin sebaceous glands. In this paper we describe the isolation of a Mup gene, Mup-1.5a, which is expressed predominantly in the submaxillary gland of BALB/c mice. We show that Mup-1.5a is a member of a subfamily consisting of two closely related genes, both of which are closely linked to the Mup-1 locus on mouse chromosome 4. Mup-1 is the locus of a class of Mup genes (Group 1) expressed in the liver. The complete nucleotide sequence of Mup-1.5a has been determined, and was compared to a previously sequenced Group 1 Mup gene. The comparison shows that the differentially expressed Mup genes are uniformly divergent in exons, introns and in their flanking sequences. The regions of homology extend at least 5 kb into the 5' flanking region of Mup genes.     

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.     

Protein pheromone expression levels predict and respond to the formation of social dominance networks

Communication signals are key regulators of social networks and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odours of dominants and nondominants differentially influence behaviour, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild‐derived mice by analysing expression levels before, during and after competition for reproductive resources over 3 days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species.     

Variation in the Major Urinary Protein Multigene Family in Wild-Derived Mice

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.     

Tissue-specific expression of the rat alpha 2u globulin gene family.

The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.