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.     

Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.     

Comparative developmental analysis of the parotid, submandibular and sublingual glands in the neonatal rat.

Analysis of the soluble protein fractions from the rat parotid, submandibular and sublingual glands by polyacrylamide-gel electrophoresis reveals similarities in overall patterns of protein synthesis at birth. Tissue-specific changes in protein and glycoprotein synthesis occur shortly after birth and again at the time of weaning, 21--28 days later. Incorporation of [3H]thymidine into DNA was at its highest after birth and gradually decreased in both the parotid and submandibular gland, whereas [3H]thymidine incorporation in the sublingual gland was low throughout the time of neonatal development. [14C]Leucine incorporation into total protein increased in all glands with age after birth, showing an accelerated rate 21--28 days later. Trichloroacetic acid/phosphotungstic acid-precipitable [3H]fucose in glycoproteins declined over the time of neonatal development in the parotid and submandibular gland, but its incorporation remained higher in the sublingual gland. alpha-Amylase (EC 3.2.1.1) in the salivary glands increased at the time of weaning, as judged by detectability in sodium dodecyl sulphate/polyacrylamide gels and by immune precipitation. Two membrane-bound enzymes, UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) and UDP-galactose:2-acetamido-2-deoxy-D-galactosaminyl-protein 3 beta-galactosyltransferase (no EC number), undergo tissue-specific change rather than changes induced by physiological stimulation of the salivary glands.     

Structural genes of the mouse major urinary protein are on chromosome 4

The major urinary proteins (MUPs) of mouse are a family of at least three major proteins which are synthesized in the liver of all strains of mice. The relative levels of synthesis of these proteins with respect to each other in the presence of testosterone is regulated by the Mup-a locus located on chromosome 4. In an effort to determine the mechanism of this regulation in molecular terms, a cDNA clone containing most of the coding region of a MUP protein has been isolated and identified by partial DNA sequence analysis. Using a combination of hybridization analysis and somatic cell genetics, the structural gene family has been unambiguously mapped to mouse chromosome 4. These data suggest that Mup-a regulation operates in a cis fashion and that models proposing trans regulation of MUP protein synthesis are unlikely.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Progesterone metabolism by major salivary glands of rat--I. Submandibular and sublingual glands

The metabolism of progesterone by the submandibular and sublingual salivary glands of female (nonpregnant and pregnant) and male rats was studied. The metabolism was in both sexes significantly greater in submandibular than in sublingual glands. Sex differences were not seen in sublingual glands but less metabolism was found in homogenates and microsomal fractions of female (nonpregnant and pregnant) submandibular glands compared to that of males. The metabolism did not differ between pregnant and nonpregnant female rats. The metabolites were mainly 5 alpha-pregnane-compounds. On the basis of the metabolites identified it can be concluded that rat submandibular and sublingual glands contain at least 3 alpha-, 3 beta-, 20 alpha- and 20 beta-hydroxysteroid dehydrogenase, 5 alpha- and 5 beta-steroid hydrogenase and 17 alpha-steroid hydroxylase activity. 5 alpha-steroid hydrogenase activity was significantly higher in all preparations of male submandibular glands than in females. In sublingual glands some enzyme activities showed pregnancy-related decreased.     

Two main groups of mouse major urinary protein genes, both largely located on chromosome 4.

Fourteen different major urinary protein (MUP) genomic clones from BALB/c mice were isolated. By restriction site mapping, six of these form two sets of three overlapping clones. By the criterion of cross-hybridization, the 10 different genes fall into two groups of four (Group 1) and three (Group 2) genes, while three genes fall into neither group. Southern blot analysis of genomic DNA with Group 1 and Group 2 plasmid subclones shows that the haploid mouse (BALB/c) genome contains approximately 15 Group 1 genes, 12 Group 2 genes and at least seven MUP genes that belong to neither group. An analysis of mouse-Chinese hamster hybrid cell lines shows that most, if not all, Group 1 and Group 2 genes are located on mouse chromosome 4.     

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3''-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.     

Differential expression in male and female mouse liver of very similar mRNAs specified by two group 1 major urinary protein genes.

The major urinary proteins of the mouse are encoded by a large multigene family composed of several distinct groups of genes distinguished by differences in sequence and expression characteristics. The genes in the largest group (group 1) show greater than 99% pairwise similarity in their exons. By hybridization between RNA and a specifically designed oligonucleotide, we confirmed that genes of this group are expressed mainly in the liver. By using additional gene-specific oligonucleotide probes, we have been able to distinguish between the species of mRNA corresponding to two of these genes and to measure their abundance in male and female liver. Both mRNAs are present in male liver at high but different levels. Both are also present in female liver, one at a much lower level than in the male and the second at a very low level indeed. Both are present at male levels in the livers of females induced with testosterone. These results show unequivocally that the expression of different group 1 Mup genes is differentially influenced by the hormonal status of the mouse.     

Malignant transformation in concurrent benign mixed tumors of the parotid and submaxillary glands

This patient is the only reported case of concomitant mixed tumors of the parotid and contralateral submaxillary gland. Each lesion had histologic changes consistent with different stages in the transformation of a benign to a malignant mixed tumor. Identifying premalignant disease in the parotid tumor and early preinvasive carcinoma in the submaxillary tumor lends support to the concept of malignant progression in salivary gland mixed tumors.