Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis

The inositol/choline responsive element (ICRE) functions as a UAS element mediating coordinate expression of structural genes required for yeast phospholipid biosynthesis. However, ICRE motifs could be detected upstream of various genes apparently not involved in lipid metabolism. In this work we investigated the expression pattern of selected genes containing ICRE promoter motifs, as identified by in silico analysis (ARG4, ERG20, FAR8, GPD2, RSF1, URA8, VHT1 and YEL073C). It turned out that the presence of an ICRE upstream of a gene of unknown function indeed allows to conclude for regulation by phospholipid precursors, which is mediated by activators Ino2/Ino4 and the repressor Opi1. We also demonstrated in vitro binding of Ino2/Ino4 heterodimers to promoter regions. Thus, our analysis supports the view that identification of regulatory elements by a database search provides evidence for a specific pattern of gene expression. Activation by pathway-specific regulators may suggest a physiological function for as yet uncharacterized genes.     

Functional inactivation of the conserved Sem1p in yeast by intrabodies

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.     

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.     

Role of the N-terminal region of Rap1p in the transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

In the yeast two-hybrid system, the N-terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two-hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N-terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N-terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N-terminal region of Rap1p. These results suggest that one function of the N-terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein-protein interaction.     

Biochemical and genetic characterization of Yra1p in budding yeast

Yra1p and its vertebrate homologues bind to the mRNA export factor Mex67p/TAP and are thought to play a role in mRNA export in vivo. To further characterize Yra1p, we used immunoaffinity chromatography to purify endogenous Yra1p complexes. These experiments demonstrated that two importin beta homologues (Kap123p and Pse1p) and the poly A tail-binding proteins Pab1p and Nab2p associate with Yra1p. The other major proteins that associate with Yra1p include proteins involved in mRNA and rRNA processing and the Yra1p-related protein Yra2p. Additional biochemical and genetic experiments suggest a close functional relationship between Yra1p and Yra2p. We generated a temperature-sensitive allele of YRA1 and used it to demonstrate that cells which lack the function of both Yra1p and Yra2p are able to exit a G0 arrest and go through several rounds of cell division before arresting. We also identified high-copy suppressors of the yra1-2 temperature-sensitive growth defect. These include SUB2, a splicing factor important in mRNA export, ULP1, a nuclear cysteine protease localized to the nuclear pore and involved in Smt3p/SUMO processing, and YRA2. Taken together, these results suggest that Yra1p has roles in diverse RNA processing events in addition to a role in mRNA export.     

天山一号冰川产果胶酶酵母菌的筛选及其系统发育分析

从前期天山冰川冻土及融水中分离到68株可培养酵母菌中,通过筛选性培养基筛选产低温果胶酶的菌株。选择培养基显示菌株C2、C9、L1和L13可产低温果胶酶。对4株酵母菌最适生长温度进行测定,16 ℃条件下4株酵母菌均能生长,属耐冷菌范畴。通过26S rDNA基因序列分析确定产果胶酶菌种的系统进化地位,在系统发育上,4株产酶菌分别与Cryptococcus macerans DQ377662、Cryptococcus sp. DQ377668、Rhodotorula laryngis JQ768911、Rhodotorula sp. JX124722的同源性最高。