Abundant androgen regulated mRNAs in mouse submandibular gland: cell-free translation of renin precursor mRNA

Submandibular glands of male mice contain at least four abundant mRNAs that occur at low concentrations in glands of females. The male-specific mRNAs code for polypeptides of 48,000, 43,000, 29,000, and 27,000 MW. Androgenic regulation of these mRNAs is illustrated by their apparent absence in glands of castrate males and by their accumulation in glands of females treated with testosterone. Selective hybrid-arrested translation experiments also indicate reduced levels of these male-specific sequences in female gland cytoplasm. The 48,000 MW male-specific polypeptide is reduced in translation products directed by gland mRNA from C57BL10/J mice (variants deficient in salivary renin), suggesting the corresponding mRNA codes for a renin precursor. The identity of this polypeptide is confirmed by immune selection with renin-specific antibody.     

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.     

Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.     

Blocking of in vitro translation of Paramecium messenger RNAs is due to messenger RNA primary structure

Paramecium primaurelia mRNAs were translated in vitro in rabbit reticulocyte lysate and the products of translation were analyzed by their size. We show that the large majority of these products are of short but discrete sizes irrespective of the length of the mRNA which directs their synthesis. An illustrative example is given by the translation of mRNA of G surface antigen which directs the synthesis of a 50 kD polypeptide instead of the complete 250 kD protein. Control experiments suggest that the blocking is due to mRNA primary structure.     

Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5' ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.     

The structural genes for three Drosophila glue proteins reside at a single polytene chromosome puff locus.

The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Cell-free translation of proline-rich protein mRNAS from human submandibular gland

RNA was isolated from a human submandibular gland and separated into poly A-enriched and poly A-deficient fractions by chromatography on oligo (dT) cellulose. Both of these RNA fractions stimulated methionine incorporation into polypeptides in a reticulocyte lysate translation system. Two in vitro translation products templated by poly A-enriched mRNA were isolated by immunoprecipitation with immune serum directed against human salivary anionic proline-rich protein I. These polypeptides were shown to be precursors of proline-rich proteins on the basis of Mr, affinity for the antiserum, and preferential incorporation of proline. This study is the first to demonstrate cell-free translation of the mRNAs for human proline-rich salivary protein precursors.     

A monoclonal antibody directed against high Mr salivary mucins recognizes the SO3-3Gal{beta}1-3GlcNAc moiety of sulfo-Lewisa: a histochemical survey of human and rat tissue

Using a panel of synthetic oligosaccharides attached to a polyacrylamidecarrier, the epitope of monoclonal antibody F2, evoked to highM_r salivary mucins, was mapped to the SO₃-3Galß1-3GlcNAc-moiety of the sulfo-Le^a antigen. Using immunochemical techniques,the expression of the F2-epitope was investigated in a numberof different isolated human mucin species, as well as in humanand rat tissue specimens. The mAb F2 bound to high M_r salivarymucins, cervical mucins, colon mucins and gallbladder mucins,but not to low M_r salivary mucins nor to gastric mucins. Immunohistochemicalscreening of human tissues with mAb F2 revealed a positive reactionwith a number of epithelia, including the (sero)mucous salivaryglands, the goblet cells of the colon, submucosal glands ofthe lung, the lining epithelium of cervical and esophageal glands,the suprabasal skin keratinocytes, and Hassall's corpusclesof the thymus. No staining was found in normal breast, pancreas,small intestine, spleen, and lymph nodes. Normal gastric glandswere negative, but gastric intestinal metaplastic glands stronglystained with the antibody. In rat tissues, mAb F2 labeled epithelialcells of salivary glands, colon and stomach. In addition toepithelial cells, extracellular matrix components in rat thymusand skin were labeled by mAb F2. No labeling of erythrocytes,granulocytes, lymphocytes or bone marrow cells was found byFACScan analysis. The present data shows a tissue specific distributionof the F2-epitope in cells from the epithelial lineage in humanand rat. epithelial tissue sulfo-Lewis^a mucins mAbs immunohistochemistry     

Cell death in the salivary glands of metamorphosing calliphora vomitoria

The salivary gland cells of Calliphora vomitoria larvae initiate and complete their own destruction in a programmed manner at the onset of metamorphosis. On entering the post-feeding period the larvae come to rest and the polytene salivary gland cells show a significant increase in DNA synthesis followed closely by a surge of mRNA synthesis accompanied by increasing protein production. During this prelude to cell death the new mRNA gives rise to at least 10 new proteins. The first new proteins having a MWt between 30 and 100kD appear by day 8 of the life-cycle and a number persist until the advent of cell death on day 9. Other new proteins appear in a cascade of production during day 8 and in vitro translation of mRNA produced at this time shows a new 55kD protein appearing before cell destruction. Significantly no evidence of DNA degeneration or laddering associated with classical apoptosis was observed, on the contrary considerable DNA synthesis in the form of chromosomal endoduplication or "genomic amplification" was seen; selective gene expression being apparently controlled at translational level. Overall the results strongly suggest a synthetically mediated programmed cell death in the metamorphosing salivary glands of the blow-fly which is distinct from apoptosis.     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

Alternative splicing model for the synthesis and secretion of the 20 kilodalton form of rat growth hormone

The characterization of a 20 kilodalton (20 kD) variant of rat growth hormone is reported. The 20 kD variant from rat pituitary gland extracts was identified on Western immunoblots of polyacrylamide gels. It was also shown that pituitary tissue maintained in culture secretes the 20 kD form. A rat growth hormone cDNA fragment was used as a probe in S1 nuclease mapping experiments of rat pituitary poly (A) mRNA to detect the presence of two growth hormone mRNAs in the rat pituitary gland. The protected mRNAs correspond to the predicted sizes that would encode the 22 kD and 20 kD forms of growth hormone. The site of variation between the mRNAs maps to a potential alternative 3' splice site in the 5' end of exon 3 of the coding sequence. The results support the hypothesis that the 20 kD variant in rat is the product of an mRNA alternatively spliced in exon 3, as is the case for the human growth hormone.     

A single mouse alpha-amylase gene specifies two different tissue-specific mRNAs

The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.     

Basic proline-rich proteins of murine parotid glands. Induction of mRNA by isoprenaline and post-secretion processing

Five major basic polypeptides with characteristics typical of proline-rich proteins, accumulated in parotid glands after long term isoprenaline treatment of Balb C mice. They were studied by two-dimensional gel electrophoresis and designated B1 degree, B2' degrees, B2 degrees, B3 degrees and B4 degrees on the basis of pI-dependent mobility. They were not observed in the glands of normal mice and were precipitated when glands were homogenized in 10% trichloroacetic acid unlike the three isoprenaline-induced proline-rich proteins of murine parotid glands reported previously. Isoprenaline induced six proline-rich in vitro translation products which were absent normally. Four of these species had pI-dependent mobilities almost identical to B1 degree, B2 degrees, B3 degrees and B4 degrees, indicating not only precursor/product relationships, but also that isoprenaline induced the accumulation of the proteins by regulating the mRNA. Identical salivary counterparts of the basic glandular proline-rich proteins were not detected whereas a series of smaller and more basic isoprenaline-induced polypeptides were observed in saliva (major speices B1s-B4s). The glandular proline-rich proteins were secreted from parotid tissue in vitro and the data indicate that proline-rich proteins are synthesised as precursors and processed into salivary form in the parotid glands after secretion. The relationships between the B-type in vitro translation products, parotid gland precursors and salivary proteins were also confirmed immunologically.     

Tissue-specific expression of mouse α-amylase genes: Nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland

We have determined the nucleotide sequence of two different mouse α-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively, are the major α-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5′ noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict α-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland α-amylase mRNA is not initiated at the AUG codon nearest the 5′ terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.