The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions

The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a?Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 prot- ects cells against stress, whilst overexpression of this gene has the opposite effect. MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA). Both activation of PKA or overexpression of SOK1 or SCH9– two genes isolated as multicopy suppressors of a PKA null mutant – suppress the mbr1 growth defect. Our results indicate that Mbr1p is not an essential element of any one of these pathways. Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality. A possible role of Mbr1p in intracellular trafficking is discussed.     

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.     

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     

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.     

The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5′ region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5′ sequences under carbon-limiting conditions.     

The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions

The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 prot- ects cells against stress, whilst overexpression of this gene has the opposite effect. MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA). Both activation of PKA or overexpression of SOK1 or SCH9– two genes isolated as multicopy suppressors of a PKA null mutant – suppress the mbr1 growth defect. Our results indicate that Mbr1p is not an essential element of any one of these pathways. Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality. A possible role of Mbr1p in intracellular trafficking is discussed. Received: 17 January 1997 / Accepted: 20 March 1997     

Genetic mapping of a leaf rust resistance gene in the former Yugoslavian barley landrace MBR1012

Leaf rust, caused by Puccinia hordei Otth, is an important disease of barley (Hordeum vulgare L.) in many areas of the world. The appearance of new virulent races necessitates the identification of new resistance genes in barley. Screening of spring barley landraces from former Yugoslavia led to the identification of an accession (MBR1012) carrying resistance to the most widespread virulent leaf rust pathotypes in Europe. Ninety-one doubled haploid lines derived from a cross between landrace MBR1012 and the susceptible German cultivar Scarlett were evaluated for resistance to P. hordei isolate I-80 and segregated 48 resistant : 43 susceptible ( $ \chi_{1:1}^{2} = \, 0.29, $ p?=?0.6), indicating a monogenic inheritance of resistance. Using simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers, the resistance gene in MBR1012 was mapped to the telomeric region of chromosome 1HS. This gene is assigned the temporary locus designation of Rph _MBR1012 until it can be unequivocally determined to be different from all previously reported resistance genes. The closest flanking markers for Rph _MBR1012 are located 0.8?cM distal (SNP marker GBS546 and SSR marker GBMS187) and 6.0?cM proximal (SSR marker GMS21). The diagnostic value of the closest linked markers was assessed in a genetically diverse collection of 51 susceptible and resistant barley lines and cultivars. The SSR GBMS187 predicted the presence of Rph _MBR1012 with 100% accuracy. However, this marker could not be used singly for the rapid incorporation of resistance into high yielding barley cultivars, since it detects a null allele in MBR1012. Therefore, simultaneous use of the markers closely linked to Rph _MBR1012 is needed for transferring Rph _MBR1012 into adapted cultivars.     

Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant

It has been reported that an indigenous quorum quenching bacterium, Rhodococcus sp. BH4, which was isolated from a real plant of membrane bioreactor (MBR) has promising potential to control biofouling in MBR. However, little is known about quorum quenching mechanisms by the strain BH4. In this study, various characteristics of strain BH4 were investigated to elucidate its behavior in more detail in the mixed liquor of MBR. The N-acyl homoserine lactone hydrolase (AHL–lactonase) gene of strain BH4 showed a high degree of identity to qsdA in Rhodococcus erythropolis W2. The LC-ESI-MS analysis of the degradation product by strain BH4 confirmed that it inactivated AHL activity by hydrolyzing the lactone bond of AHL. It degraded a wide range of N-acyl homoserine lactones (AHLs), but there was a large difference in the degradation rate of each AHL compared to other reported AHL–lactonase-producing strains belonging to Rhodococcus genus. Its quorum quenching activity was confirmed not only in the Luria-Bertani medium, but also in the synthetic wastewater. Furthermore, the amount of strain BH4 encapsulated in the vessel as well as the material of the vessel substantially affected the quorum quenching activity of strain BH4, which provides useful information, particularly for the biofouling control in a real MBR plant from an engineering point of view.     

Identification,characterization and interaction of <Emphasis Type="Italic">HAP</Emphasis> family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB288027 to AB288048 and BR000373 to BR000375.