Mucin genes are regulated by estrogen and dexamethasone.

This study demonstrates for the first time that mucin gene expression can be significantly up-regulated by steroid hormones. The effects of estrogen, progesterone and dexamethasone on the expression of three mucin genes, MUC1, MUC4 and MUC5c were determined in a human epithelial cell line. This cell line secretes mucin and contains steroid receptors. Steady-state mRNA levels were quantified by slot blot hybridization using radiolabelled oligonucleotide probes. Normally, these genes (originally isolated from airways) are expressed at low constitutive levels. However, steady state levels of MUC4 and MUC5c mRNA were up-regulated at least 3-4 fold by estrogen supplementation, and 1.5-2 fold by dexamethasone. MUC1 mRNA levels were minimally affected. Progesterone supplementation had no effect but was able to interfere with the up-regulation by estrogen. The mechanism of regulation remains to be determined but may be transcriptional. However, steroids have been shown to control gene expression at several different levels.

Regulation of mucin gene expression in secretory epithelial cells.

To gain insight into the regulation of mucin biosynthesis and secretion at a molecular level, we studied the expression of four mucin genes in several secretory cell types. Oligonucleotide probes for MUC1, MUC2, MUC3, and MUC4 were used in Northern blot analysis of total RNA from the cells. It was shown that MUC4 was constitutively expressed in the endometrial cell line (Ishikawa), which we use as a model system for studying airway mucin. Expression was enhanced by secretagogues. Cultured bronchial epithelial cells gave identical results. MUC1, MUC2, and MUC3 were not detected in either cell system. Secretory phase endometrial tissues also expressed only MUC4, but proliferative tissues expressed both MUC4 and MUC1. The data indicate that the mucin genes are differentially expressed in various cells and suggest a possible regulatory role for steroid hormones.

Isolation and characterization of a macrophage-derived high molecular weight protein involved in the regulation of mucus-like glycoconjugate secretion.

Pulmonary macrophages release a variety of mediators that are involved in inflammatory processes and probably are involved in respiratory mucus secretion. Conditioned media obtained from activated pulmonary macrophages were found to contain a protein that functioned as a secretagogue for mucus-like glycoconjugate (MLGC) in an in vitro bioassay. A human macrophage-derived hybridoma cell line, HB-63, exhibited the same properties and was very useful in obtaining large amounts of the protein for purification and characterization. With ultrafiltration membranes and gel electrophoresis, the protein isolated from the conditioned media of zymosan- or lipopolysaccharide-treated cells was found to have a molecular weight of approximately 68 kd. The purified protein obtained from hybridoma cells and from pulmonary macrophages exhibited strong biologic activity when it was used to stimulate MLGC secretion, both in human airway explants and in an in vitro human secretory epithelial cancer cell line. The proteins from both sources were found to have similar amino acid compositions. Preliminary results indicate the presence of the 68 kd protein in the bronchoalveolar lavage fluid of a patient with severe chronic bronchitis and mucus hypersecretion. The role of this novel protein in the lungs is, so far, speculative. The 68 kd protein may be a useful tool for studying the biosynthesis and regulation of MLGC secretion and hypersecretion.

Regulation by heme of the synthesis of cytochrome C oxidase subunits V and VII in yeast.

The effects of heme on the synthesis of subunits V and VII of cytochrome c oxidase were examined in the heme-deficient yeast mutant, GL1. In vitro translation and immunodetection with subunit-specific antisera indicated a 50% decrease in both proteins, with RNA obtained from hemeless cells. Unsupplemented, pulse-labeled cells contained both V and VII polypeptides, but at extremely low levels as compared with those found in delta-aminolevulinic acid-supplemented cells. The data suggest that heme controls the formation of mRNAs for the two subunits, and may also have a regulatory role in translation and in the stability of the polypeptides.

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells.

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.

Synthesis of the apoprotein of cytochrome b in heme-deficient yeast cells.

The presence of the apoprotein of cytochrome b has been demonstrated in a mutant of Sacchromyces cerevisiae lacking delta-aminolevulinic acid synthase and, hence, devoid of heme. The apoprotein of cytochrome b present in the mutant was identical with cytochrome b of control cells (mutant cells grown in the presence of delta-aminolevulinic acid) by the following criteria: similar apparent molecular weights in dodecyl sulfate-polyacrylamide gel electrophoresis; anomalous migration behavior during electrophoresis in polyacrylamide gels of different porosities; identical gel pattern obtained after immunoprecipitation with specific antiserum against cytochrome b; and identical fingerprints obtained after limited proteolysis with Staphylococcus aureus V8 protease. The kinetics of incorporation in vivo of [35S]methionine into apoprotein of cytochrome b in the mutant suggested that heme deficiency may affect assembly into the membrane of subunits of the cytochrome b.c1 complex rather than synthesis of cytochrome b.

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.

tyrR, a regulatory gene of tyrosine biosynthesis in Salmonella typhimurium.

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which tyrosine pathway enzymes were not repressed by l-tyrosine. The mutants produced elevated levels of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, and these enzymes as well as transaminase A were not repressed by high concentrations of tyrosine. Genetic analysis revealed that a mutation in a gene designated tyrR was responsible for the constitutivity of the tyrosine pathway enzymes in strains SG1, SG7, and SG9, and that tyrR was linked to pyrF. In strain SG1 a mutation had also occurred in aroF, the structural gene for DAHP synthetase (tyr), resulting in loss of sensitivity of this enzyme to end-product inhibition. There appeared to be no relationship between loss of feedback inhibition and loss of end-product repression, since derivative strains of SG1 that carried only the tyrR mutation behaved like the singly mutated tyrR strains, SG7 and SG9, in showing high constitutive levels of tyrosine-specific enzymes that were not repressed by tyrosine.

Regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium.

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase (specified by pheA) was highly elevated. Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA, and conjugation and merodiploid analysis indicated that it was in the 95- to 100-min region of the Salmonella chromosome. Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR, a gene specifying a cytoplasmic product that affected pheA. pheR mutants were found to carry a second mutation, tyrO. The tyrO mutation acts cis to cause increased levels of the tyrosine biosynthetic enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthetase (tyr) and prephenate dehydrogenase, but it has no effect on regulation of pheA.