Upregulation of low density lipoprotein receptor by gemfibrozil, a hypolipidemic agent, in human hepatoma cells through stabilization of mRNA transcripts.

Gemfibrozil reduces the plasmal levels of cholesterol and triglyceride in patients with hyperlipidemia by a mechanism that is not well understood. The present study evaluated the effect of gemfibrozil on the LDL receptor in human hepatoma cells compared with that of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Exposure to gemfibrozil, 40 mumol/L, for 3 days increased the binding of 125I-LDL to the surface of three lines of human hepatoma cell, HepG2, HuH7, and HLE by 1.5- to 2.0-fold. Similar findings were observed with pravastatin. Scatchard analysis with 125I-LDL indicated an increased number of LDL receptors on the cell surface of HepG2 cells when treated with gemfibrozil and pravastatin. However, the gemfibrozil-treated cells exhibited no increase in the binding of 125I-epidermal growth factor (EGF). Gemfibrozil increased the levels of LDL receptor mRNA and protein in HepG2 cells. The increase in LDL receptor activity induced by pravastatin was abolished by concomitant administration of mevalonic acid, 770 mumol/L. This effect was not seen with gemfibrozil, suggesting the mechanism differs for the two lipid-lowering drugs. To determine whether this increase in mRNA was due to transcriptional activation, we prepared HepG2 cells transfected with an LDL receptor promoter-reporter construct that contained a sterol regulatory element. The expression of LDL receptor regulated by the sterol regulatory element was increased by pravastatin, but not by gemfibrozil. We evaluated the stability of the mRNA in the presence of actinomycin D to explain the increase in the LDL receptor mRNA. Gemfibrozil prolonged the half-life of the mRNA for LDL receptor but not that for the EGF receptor. Stabilization of the LDL receptor mRNA is suggested to be the novel mode of action of gemfibrozil.

Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes.

We examined the effect of pravastatin sodium (pravastatin), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on fatty acid synthesis in rat liver. The repeated administration of pravastatin to rats at 250 mg/kg for 7 days led to a 2.8-fold increase in fatty acid synthesis in the liver. The diurnal change of fatty acid synthesis was not affected by the treatment. Hepatic fatty acid synthase activity was increased 3.2-fold, while acetyl-CoA carboxylase activity was not changed by the repeated administration of pravastatin. In rat hepatocytes, the incubation with 2 microg/ml pravastatin for 24 h increased fatty acid synthase activity 1.5-fold, as well as HMG-CoA reductase activity 2.8-fold. These results suggest that HMG-CoA reductase inhibitors might increase fatty acid synthesis in vivo through the induction of hepatic fatty acid synthase.

The mechanism of comparable serum cholesterol lowering effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A inhibitor, between once- and twice-daily treatment regimens in beagle dogs and rabbits.

In dogs, no significant difference in the reduction of serum cholesterol was observed among three dosing regimens of pravastatin: once in the morning (3 mg/kg), once in the evening (3 mg/kg), and twice-daily (1.5 mg/kg x 2) for 21 days. In rabbits, pravastatin was administered at a dose of 50 mg/kg once-daily given in the evening or 25 mg/kg twice-daily for 14 days; the respective serum and liver cholesterol levels were decreased by 41% and 7% in the once-daily dosing and by 51% and 11% in the twice-daily dosing. The amount of low density lipoprotein (LDL) receptor protein was increased 1.2-1.3-fold (P < 0.05) by both treatments, and no significant difference was noted between these treatment regimens. In addition, there was no significant difference in the extent of up-regulated LDL receptor protein between once-daily dosing in the evening and once-daily dosing in the morning. In the experiments with rabbit hepatocytes, the up-regulated LDL receptor activity induced by preincubation with pravastatin was retained even 24 hr after the removal of pravastatin. These results suggest that the comparable efficacy of pravastatin between once- and twice-daily treatment could be explained by retention of up-regulated LDL receptor activity for more than 24 hr in vitro and in vivo.

Characterization and mapping of the Bacillus subtilis prtR gene.

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.

Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes.

In Bacillus subtilis, the extracellular enzyme levansurcrase is synthesized in the presence of sucrose. A termination structure between the transcription start site and the structural gene was the apparent site for regulation by sucrose of transcription into the structural gene. Sequence analysis of the sacB leader region from two strains constitutive for levansucrase synthesis showed a single base change in the stem of this termination structure. This single base change also led to the constitutive synthesis of a sacB'-'lacZ fusion, whereas the wild-type sacB'-'lacZ fusion was induced by the addition of sucrose. S1 nuclease mapping of sacB transcripts with probes labeled either within the termination structure or 3' to the termination structure showed that sucrose did not increase the number of transcripts extending into the termination structure; however, sucrose did increase the number of transcripts extending past the termination structure. Two pleiotropic mutations which affect the expression of levansucrase, sacQ36 hyperproducing [sacQ36(Hy)] and sacU32(Hy), were separately introduced into the strain carrying the sacB'-'lacZ fusion. These mutations each increased the expression levels of the sacB'-'lacZ fusion. S1 mapping showed increased levels of transcript initiating at the sacB promoter in strains with the sacQ36(Hy) and sacU32(Hy) mutations. This increased transcription appeared to be independent of the sucrose-regulated transcription termination, suggesting the existence of at least two different mechanisms for the regulation of sacB expression.

Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons.

The trp operon regulatory region of Bacillus pumilus was cloned and sequenced. The cloned B. pumilus trp promoter-leader region functioned in Bacillus subtilis to express the adjacent leukocyte interferon A gene on a multicopy transcriptional fusion plasmid, pBpIFI. In strains carrying this plasmid, anthranilate synthetase levels were elevated, possible due to titration of a B. subtilis trp regulatory factor by multiple copies of the transcript of the plasmid-borne B. pumilus trp leader region. The B. pumilus trp promoter was recognized efficiently in vitro by B. subtilis sigma 43 RNA polymerase. Approximately 12% of the transcripts produced in vitro terminated in the leader region immediately following synthesis of a transcript structure resembling rho-independent terminators of enteric bacteria. An analogous terminator exists in the B. subtilis trp leader transcript. Nucleotide sequence comparison of the B. pumilus and B. subtilis trp leader regions revealed conservation of these and other sequences that could form transcript secondary structures postulated to regulate transcription termination in B. subtilis (H. Shimotsu, M.I. Kuroda, C. Yanofsky, and D.J. Henner, J. Bacteriol. 166:461-471, 1986). We propose that two elements implicated in B. subtilis trp operon regulation are conserved in the related organism B. pumilus: alternative transcription antiterminator and terminator structures in the leader transcript, and a trans-acting factor present in limiting amounts that is required for transcription termination in the leader region.

Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon.

Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis.

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.

Nucleotide sequence of the Bacillus subtilis tryptophan operon.

In Bacillus subtilis, tryptophan biosynthesis is one of the most thoroughly characterized biosynthetic pathways. Recombinant DNA methodology has permitted a rapid characterization of the tryptophan (trp) gene cluster at the molecular level. In this report the nucleotide sequence of the six structural genes together with the intercistronic regions and flanking regulatory regions are presented.

Characterization of the Bacillus subtilis tryptophan promoter region.

The nucleotide sequence of the control region of the trp operon of Bacillus subtilis has been determined. The region was shown to contain the trp promoter by deletion analysis and by determination of the transcription start site. The trp promoter shows similarity to the consensus sequence for Escherichia coli and B. subtilis promoters. The presence of the trp control region on a high-copy-number plasmid confers resistance to the tryptophan analogue 5-methyltryptophan. It appears that an approximately 120-base-pair region comprising not only the trp promoter but also adjacent direct repeat sequences is necessary to confer 5-methyltryptophan resistance. We postulate that this region is involved in tryptophan regulation and confers 5-methyltryptophan resistance by titration of a trp regulatory protein. Removal of either the trp promoter or the adjacent direct repeat sequences abolished the 5-methyltryptophan-resistance phenotype. Placement of unrelated promoters adjacent to the direct repeat sequences restored 5-methyltryptophan resistance. This suggests that promoter activity is necessary for the regulatory function.

Cloning of sporulation gene spoIIC in Bacillus subtilis.

Specialized transducing phages rho 11spoIIC and phi 105spoIIC, carrying the Bacillus subtilis sporulation gene spoIIC, were constructed by the prophage transformation method. An EcoRI fragment (2.4 MDal) carrying the spoIIC gene was isolated from the phi 105spoIIC genome and recloned into the EcoRI site of plasmid pUB110. The recombinant plasmids corrected the sporulation defect of a Spo- Rec- host, but slightly inhibited the sporulation of a Spo+ Rec- host.

Isolation and characterization of a pock-forming plasmid pTA4001 from Streptomyces lavendulae.

A covalently closed circular (ccc) DNA with a size of 5.9 kb was isolated from a strain producing streptothricins , Streptomyces lavendulae No. 1080, and its restriction map was determined. Colonies of the plasmid-carrying strain formed pocks on the lawn of the plasmid-free derivatives of the same organism. The pock-forming ability of pTA4001 was confirmed by transformation of S. lividans with the plasmid DNA. Insertion of the chromosomal streptothricin resistance gene of S. lavendulae No. 1080 into pTA4001 gave various composite plasmids with marked deletion in S. lividans as a host. By analyzing these derivatives, a 2.2 kb region essential for replication and a possible region for pock formation were determined in the map of pTA4001 . A cloning vector, pKST2 (4.3 kb) containing resistance genes to streptothricin and thiostrepton with several unique cloning sites was constructed from pTA4001 .

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes.

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.

Selection for restriction-induced in vivo deletion in phage vector phi 1E1 of Bacillus subtilis.

Recombinant phage phi 1E1metB, which contains the 4.5-kb EcoRI fragment of Bacillus subtilis DNA, has no HaeIII cleavage sites within the vector phi 1E1 genome but only in the metB insert. When phi 1E1metB was grown in B. subtilis ISR11, which produces BsuR, the isoschizomer of HaeIII, it was restricted and survived with an efficiency of approx. 10(-5). All the survivors were deletion mutants of phi 1E1metB, and only various segments of the insert DNA delineated by HaeIII sites were deleted. The Met+ transforming activities of the DNAs from phi 1E1metB and its deletion derivatives were examined, and the restriction maps of the deletion mutants were correlated with five metB- mutation sites.

Early sporulation gene spo0F: nucleotide sequence and analysis of gene product.

We have determined the sequence of a 1,162-base-pair DNA fragment containing a spo0F gene which is required for an early stage of sporulation in Bacillus subtilis. The sequence has only one long open reading frame consisting of 173 codons, which has been confirmed to be the spo0F cistron by DNA-mediated transformation and in vitro transcription. In UV-irradiated "maxicells" containing pUBSF13, the plasmid that carries cloned spo0F gene, we have observed the synthesis of a 20-kilodalton polypeptide that is absent from cells carrying a vector plasmid pUB110. The molecular weight of this protein is in agreement with the calculated molecular weight of the spo0F gene product (Mr, 19,065). The putative promoter sequences of spo0F gene were 5' T-A-T-A-A-T 3' at -10 and 5' T-T-G-A-T-T 3' at -35. An octamer sequence, 5' A-A-A-G-G-A-G-G 3', situated 8 base pairs prior to the initiation codon was found to be perfectly complementary with the 3' end of 16S ribosomal RNA. This result offers additional evidence for the proposal by Rabinowitz's group that an extensive mRNA-rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes.

Unusually infrequent cleavage with several endonucleases and physical map construction of Bacillus subtilis bacteriophage phi 1 DNA.

HaeIII, BalI, StuI, BamHI, SlaI, and EcoRII did not cut the genome of Bacillus subtilis phage phi 1 at all, whereas ThaI, BglII, EcoRI, SalI, and Bsu1247I cut the genome once or twice. The physical map of the phi 1 genome was constructed with the latter restriction endonucleases.

A new site-specific endonuclease StuI from Streptomyces tubercidicus.

A new sequence-specific endonuclease, StuI, produced by Streptomyces tubercidicus KCC S-0054, was identified and partially purified. StuI recognizes the hexanucleotide "palindromic" sequence (Formula: see text), and cleaves it at the middle, producing blunt ends.