UBI4, the polyubiquitin gene of Saccharomyces cerevisiae , is a heat shock gene that is also subject to catabolite derepression control

 Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position −542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress. Received: 3 June 1996 / Accepted: 20 August 1996     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is a heat shock gene that is also subject to catabolite derepression control

Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position ?542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress.     

Bovine RNase MRP cleaves the divergent bovine mitochondrial RNA sequence at the displacement-loop region

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves mitochondrial RNA from the origin of leading-strand DNA synthesis contained within the displacement-loop region. Bovine mitochondrial DNA maintains the typical gene content and order of mammalian mitochondrial DNAs but differs in the nature of sequence conservation within this displacement-loop regulatory region. This markedly different sequence arrangement raises the issue of the degree to which a bovine RNase MRP would reflect the physical and functional properties ascribed to the enzymes previously characterized from mouse and human. We find that bovine RNase MRP exists as a ribonucleoprotein, with an RNA component of 279 nucleotides that is homologous to that of mouse or human RNase MRP RNA. Characterization of the nuclear gene for bovine RNase MRP RNA showed conservation of sequence extending 5 of the RNase MRP RNA coding sequence, including the presence of a cis-acting element known to be important for the expression of some mitochondrial protein-coding nuclear genes. Bovine or mouse RNase MRP cleaves a standard mouse mitochondrial RNA substrate in the same manner; each also cleaves a bovine mitochondrial RNA substrate identically. Since bovine and mouse RNase MRPs process both bovine and mouse substrates, we conclude that the structural features of the mitochondrial RNA substrate required for enzymatic cleavage have been well conserved despite significant overall primary sequence divergence. Inspection of the bovine RNA substrate reveals conservation of only the most critical portion of the primary sequence as indicated by earlier studies with mouse and human RNase MRPs. Interestingly, a principal cleavage site in the bovine mitochondrial RNA substrate is downstream of the promoter located at the leading-strand mitochondrial DNA replication origin. Correspondence to: D.J. Dairaghi     

Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

Summary The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.Abbreviations AC L-azetidine-2-carboxylic acid - Ap ampicillin - CAP catabolite gene activator protein - NR_I nitrogen regulator I - RF DNA DNA replicative form - Str streptomycin - Tc tetracycline - TTC 2,3,5-triphenyl tetrazolium chloride - UV ultraviolet     

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Summary The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Yeast HAP1 activator binds to two upstream activation sites of different sequence

We show that the HAP1 protein binds in vitro to the upstream activation site (UAS) of the yeast CYC7 gene. Strikingly, this sequence bears no obvious similarity to the sequence bound by HAP1 at UAS1 of the CYC1 gene. The CYC1 and CYC7 sites compete for binding to HAP1 and have comparable affinities for the protein. The gross features of the interaction of HAP1 with the two sites are similar: multiple major and minor groove contacts, spanning 23 bp, on one helical face, with a back-side major groove contact toward one end. The precise positions of the contacts differ, however. A mutant form of HAP1, HAP1-18, abolishes the ability of the protein to bind to UAS1 but not CYC7 DNA. Possible mechanisms for how a single protein recognizes two sequences are discussed.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.