Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription.

Different roles for abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene.

In vivo mutational analysis of the yeast RPS28A ribosomal protein (rp-)gene promoter demonstrated that both the Abf1p binding site and the adjacent T-rich element are essential for efficient transcription. In vivo Mnase and DNaseI digestion showed that the RPS28A promoter contains a 50-60 bp long nucleosome-free region directly downstream from the Abf1p binding site, followed by an ordered array of nucleosomes. Mutating either the Abf1p binding site or the T-rich element has dramatic, but different, effects on the local chromatin structure. Failure to bind Abf1p appears to cause nucleosome positioning to become disorganized as concluded from the complete disappearance of Mnase hypersensitive sites. On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by approximately 50 bp towards the Abf1p site, resulting in loss of the nucleosome-free region downstream of Abf1p. We conclude that Abf1p is a strong organizer of local chromatin structure that appears to act as a nucleosomal boundary factor requiring the downstream T-rich element to create a nucleosome-free region.

Cellular site of synthesis of ribosomal proteins in yeast.

Looking for messenger RNA coding for yeast ribosomal protein, we devised a method to identify polysomes involved in ribosomal protein synthesis. Analysis of nascent protein elongated in vitro demonstrated that ribosomal proteins are synthesized both on membrane-associated and free polysomes.

Multifunctional DNA-binding proteins mediate concerted transcription activation of yeast ribosomal protein genes.

Transcription activation of ribosomal protein genes (rp genes) in yeast is mediated through two different abundant transacting proteins, RAP1 and ABF1. These factors are multifunctional proteins playing a part in diverse cellular processes, all related to cellular growth.

Functional analysis of the promoter of the gene encoding the acidic ribosomal protein L45 in yeast.

The gene encoding the acidic ribosomal protein L45 in yeast is expressed coordinately with other rp-genes. The promoter region of this gene harbours binding sites for CP1 and ABF1. We demonstrate that the CP1-site is not involved in the transcription activation of the L45-gene. Rather, the ABF1-site, through deviating from the consensus sequence (RTARY3N3ACG), appears to be essential for efficient transcription. Replacement of this site by a consensus RAP1-binding site (an RPG box) did not alter the transcriptional yield of the L45-gene. An additional transcription activating region is present downstream of the ABF1-site. The relevant nucleotide sequence, which is repeated in the L45-gene promoter, gives rise to complex formation with a yeast protein extract in a bandshift assay. The results indicate that the L45-gene promoter has a complex architecture.

Candidate osmosensors from Candida utilis and Kluyveromyces lactis: structural and functional homology to the Sho1p putative osmosensor from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, increases in external osmolarity evoke osmostress-induced signalling via the HOG MAP kinase pathway. One of the upstream components of this signal transduction route is the putative osmosensor, Sho1p. With the aim to elucidate the molecular basis of osmosensing in budding yeast, we have cloned SHO1 homologues from Candida utilis and Kluyveromyces lactis which allowed determination of conserved domains of Sho1p. Results obtained from sequence comparisons, confirmed the importance of the transmembrane domains and the SH3 domain for Sho1p function. The K. lactis and S. cerevisiae Sho1p show the highest degree of homology, the isoform from C. utilis is a shorter protein. SHO1 from C. utilis, however, did complement the osmosensitivity of the sho1ssk2ssk22 strain by restoring HOG pathway function, since Hog1p dual phosphorylation after high osmotic challenge was restored in this strain after transformation with a plasmid bearing this SHO1 homologue.

Structure of the ABF1-homologue from Kluyveromyces marxianus.

By transformation of a Saccharomyces cerevisiae mutant strain conditionally expressing the ABF1-gene, a Kluyveromyces marxianus DNA fragment carrying the gene encoding the ABF1-homologue of this yeast strain (KmABF1) was selected. Comparison of the sequence of the KmABF1 gene with that encoding Saccharomyces cerevisiae ABF1 and the previously isolated ABF1-gene from Kluyveromyces lactis (KlABF1) revealed distinct regions displaying considerable homology and therefore most likely representing sequences encoding essential domains. In addition to the domains putatively involved in DNA binding of the protein factor, two short conserved amino acid sequence elements at the C-termini of the homologous proteins were identified, which are proposed to play a part in their trans-acting functions. This is the first report on the structure of a regulatory protein factor from K. marxianus.

The human amylase-encoding genes amy2 and amy3 are identical to AMY2A and AMY2B.

Inspection of the published nucleotide sequences reveals that the human amylase-encoding genes, amy2 and amy3, must be identical to the genes AMY2A and AMY2B, respectively.

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis.

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.

Cloning and sequencing of rhesus monkey pepsinogen A cDNA.

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Transcriptional regulation of the gene encoding cholesterol 7 alpha-hydroxylase in the rat.

The cytochrome P450 enzyme, cholesterol 7 alpha-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.

Ribosomal protein genes of yeast contain intervening sequences.

From a colony bank of HindIII-generated yeast DNA fragments we have isolated a number of recombinant DNAs carrying genes for ribosomal proteins (e.g., S10, S16A, S20, S24, S31, S33, L16, L25 and L34) of the yeast Saccharomyces carlsbergensis. By electron microscopic analysis of the R-loops formed between various DNA fragments and yeast mRNA, we could locate the ribosomal protein genes on the physical maps of the cloned DNA fragments. The R-loop structures observed indicate that a number of the ribosomal protein genes contain an intervening sequence.

The primary structure of the gene encoding yeast ribosomal protein L16.

As part of our studies on the molecular basis for the coordinate expression of ribosomal protein genes in yeast we analyzed the primary structure of the gene encoding protein L16 of the large ribosomal subunit including the flanking sequences. L16 turned out to be a ribosomal protein with a molecular mass of 22662 Da and a net charge of +12. Both the 5'- and the 3'-end of the L16 mRNA were mapped by primer extension and S1 nuclease analysis. In the DNA regions flanking the coding sequence several conserved elements are present that may be involved in transcription initiation or termination.

Nutritional upshift response of ribosomal protein gene transcription in Saccharomyces cerevisiae.

Switching Saccharomyces cerevisiae from non-fermentative to fermentative growth by adding glucose to a medium with glycerol as the sole carbon source, leads to a sudden increase in the rate of ribosomal protein gene transcription. By analyzing the nutritional shift response in a variety of yeast mutants and in the presence of different drugs, evidence was obtained that: (i) no de novo protein synthesis is required for this response; (ii) protein kinase A is essential, though independent of intracellular levels of cAMP, whereas protein kinase C is not involved; (iii) proper regulation of sugar phosphorylation is essential; (iv) glycolysis is required for the long term effect of the nutritional upshift; and (v) pathways leading to glucose-induced activation differ from those leading to gene repression, probably already at the level of glucose transport.

Analysis of the promoter of the cytochrome P-450 2B2 gene in the rat.

About 3 kb of the promoter region of the gene encoding cytochrome P-450 2B2 (CYP2B2) in the rat were sequenced and searched for potential cis-acting elements. Apart from putative binding sites for (liver-specific) protein factors, a region showing homology with the LINE 1 retrotransposon element was also found. Three proximal promoter fragments, encompassing nucleotides -579 to -372, -372 to -211, and -211 to +1, respectively, were shown to contain binding sites for multiple protein factors by bandshift analyses. The strongest protein-binding element, designated BRE (basic regulatory element), occurs between -103 to -66. Its structure is very similar to a negative control element in the murine cmyc promoter and displays a composite feature having a tandemly repeated sequence homology with the BTE (basic transcription element; Yanagida et al., 1990) separated by a CCAAA-box. The use of a deletion series of this template in in vitro transcription assays, provided evidence that the BRE serves as a major cis-acting element in the (regulated) transcription activation of the CYP2B2 gene.