Differential transcriptional regulation of the apoAI gene by retinoic acid receptor homo- and heterodimers in yeast.

Several members of the nuclear receptor superfamily including RXR (retinoid X receptor) bind to a specific retinoic acid response element (site A) of the apoAI promoter. However, transcriptional activation of the apoAI gene by different homo- and heterodimeric forms of RXR or RAR (retinoic acid receptors) cannot be evaluated in mammalian cells, which contain endogenous RXR or RAR. In order to circumvent this limitation, we assessed the DNA-binding activities and transcriptional activation of different homo- and heterodimers of these receptors in yeast. Electrophoretic mobility shift assays (EMSA) demonstrated that yeast expressed RARalpha does not bind to site A of the apoAl promoter, whereas binding of RARbeta to site A is ligand-dependent. Both RARalpha and RARbeta form heterodimers with RXRalpha and bind to site A with high affinity. These DNA-binding studies correlate with the transcriptional data, which indicated that RARbeta but not RARalpha activates transcription from site A in response equally well to 9-cis and all-trans retinoic acids. 9-cis RA is a more potent ligand than all-trans RA to activate transcription via RXR/RAR heterodimers. We conclude that this yeast expression system is a useful tool to elucidate the 'transactivation code' for apoAl site A via specific combinations of different homo and heterodimeric versions of RXR and RAR.

Cloning, expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis.

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a relative molecular mass of 25,000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein.

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli.

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

Differential transcription of the bla regulatory region during induction of beta-lactamase in Bacillus licheniformis.

Induction of beta-lactamase (blaP) in Bacillus licheniformis involves the regulatory genes blaI (repressor), blaR1 (coinducer) and R2 (function unknown). Transcription of the bla genes during induction was followed by Northern hybridization. In the first 30 min 2.3-kb transcripts encoding blaI and blaR1 were present. Subsequently, blaP mRNA and short transcripts encoding only blaI accumulated and reached a peak at 1 h. All bla transcripts turn over rapidly. Active repressor is not required for the burst of blaI-blaR1 mRNA. The production of blaI-blaR1 mRNA, and thus of BlaR1, is probably controlled both at initiation of transcription and at a later step in its synthesis and degradation.

Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis.

The expression of the blaP gene for the beta-lactamase of Bacillus licheniformis was examined by transcriptional analyses. Radiolabeled probes containing the blaP gene or various regions 3' or 5' to it were used to analyze RNA samples prepared from induced and uninduced cultures of wild-type and mutant B. licheniformis strains. The level of blaP mRNA was low in uninduced wild-type cells. At 37 degrees C, blaP mRNA levels reached a maximum 1 h after induction while rising up to 180-fold and then declined, but remained severalfold above the uninduced level for several hours. The rate of beta-lactamase synthesis was roughly proportional to the levels of blaP mRNA in both wild-type and mutant strains, indicating that regulation of beta-lactamase formation occurs primarily at the level of transcription. Turnover of blaP mRNA in the presence of rifampin was rapid, giving a blaP mRNA half-life of about 2 min. Yet, high levels of blaP mRNA were maintained for at least 1 h after removal of free inducer. Three blaP mRNAs of 1.2, 2.9, and 3.4 kilobases were produced from the blaP promoter. The most abundant made up about 97% of all blaP transcripts and was also the smallest, ending at a transcriptional terminator located about 60 bases 3' to the blaP structural gene. Variables such as incubation temperature, cytotoxicity of inducer, and type of strain had essentially no effect on the ratio of large blaP mRNA to total blaP mRNA. The 2.9- and 3.4-kilobase blaP mRNAs identify potential locations of genetically linked regulators of beta-lactamase synthesis.

Tissue distribution and excretion of 2,4-[14C]toluenediamine in the mouse.

The tissue distribution and excretion of 2,4-[14C]toluenediamine was studied in male mice given a single ip dose (1 microCi, 0.667 mg/kg). By 24 h 52% of the administered radioactivity had been excreted in the urine and 22% in the feces. The organs with the highest concentrations of radioactivity were the liver and kidneys. High concentrations of radioactivity were also observed in the gastrointestinal tract. Elimination of radioactivity from the liver, kidneys, and blood was biphasic, with half-lives of 11.7, 9.1, and 12.6 h, respectively, for the slow phases. The dominant route of excretion was via the kidneys; during the first hour after dosing, nearly 50% of the administered radioactivity was recovered in the urine. However, only an additional 2-4% of the dose appeared in the urine during the remaining 23 h of the experiment. By 24 h, only 1.25% of the administered radioactivity has been trapped from the air expired by the animals.

In vitro mutagenicity and cell-transformation screening of N-(2,3-epoxy-propyl)-phthalimide.

N-(2,3-Epoxy-propyl)-phthalimide (EPP) was tested for genetic activity in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535, TA1537 and TA100 with and without rat S9. It was inactive in strain TA1538, and active without rat S9 in TA98 at the high dose. EPP induced 6-thioguanine-resistant mutants of Chinese hamster ovary cells in the absence of an exogenous activating system. EPP produced dose-dependent enhancement of SA7 virus transformation of primary hamster-embryo cells, and transformed secondary hamster-embryo cells in a non-dose-related fashion. At a dose of 5 g/kg p.o. or i.m., EPP was inactive in the host-mediated assay using C57Bl/6XC3H mice and S. typhimurium strain TA1535. Murine testicular DNA synthesis was not inhibited by oral administration of EPP at 1000 mg/kg.

In vitro mutagenicity and cell transformation screening of phenylglycidyl ether.

Phenylglycidyl ether (1,2-epoxy-3-phenoxy propane) (PGE) was tested for genetic activity in bacterial and mammalian tests. It was active in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535 and TA100 with and without rat S9, but not in strains TA98, TA1537, or TA1538. These results suggest PGE, is a direct-acting mutagen causing base substitutions. Phenylglycidyl ether did not induce 6-thioguanine-resistant mutants of Chinese hamster ovary cells, with or without rat S9, and with or without serum in the medium. Dose-dependent enhancement of SA7 virus transformation of primary hamster embryo cells was observed at concentrations of 1.6 microgram/ml and higher. In addition, this compound was able to chemically transform secondary hamster embryo cells at concentrations of 6.2 micrograms/ml and higher. At a dose of 2500 mg/kg p.o., PGE was active in the host-mediated assay using C57B1/6 X C3H mice and S. typhimurium strain TA1535. This activity represented a positive response in 2 of 5 animals tested. Murine testicular DNA synthesis was not inhibited by oral administration of PGE at 500 mg/kg.