scholarly journals Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae

2010 ◽  
Vol 87 (4) ◽  
pp. 1327-1334 ◽  
Author(s):  
Chikara Ohto ◽  
Masayoshi Muramatsu ◽  
Shusei Obata ◽  
Eiji Sakuradani ◽  
Sakayu Shimizu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Prenyl Diphosphate ◽  
Prenyl Diphosphate Synthase

scholarly journals Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (12) ◽  
pp. 6875-6883 ◽  
Author(s):  
D J Katzmann ◽  
T C Hallstrom ◽  
M Voet ◽  
W Wysock ◽  
J Golin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Sequence Similarity ◽  
Yeast Cells ◽  
Atp Binding ◽  
Zinc Cluster ◽  
Atp Binding Cassette Transporter ◽  
Transporter Family ◽  
Encoding Gene ◽  
Atp Binding Cassette

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

scholarly journals The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 2994-3004 ◽  
Author(s):  
M Kaouass ◽  
M Audette ◽  
D Ramotar ◽  
S Verma ◽  
D De Montigny ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Fusion Gene ◽  
Transport Systems ◽  
Coding Region ◽  
Kinase Gene ◽  
Lower Affinity ◽  
High Affinity ◽  
Polyamine Transport ◽  
Exogenous Spermidine

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences

1986 ◽  
Vol 6 (12) ◽  
pp. 4335-4343
Author(s):  
J E Ogden ◽  
C Stanway ◽  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Human Interferon ◽  
Coding Region ◽  
Kinase Gene ◽  
Upstream Activation Sequence ◽  
Efficient Expression ◽  
Activation Sequence

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes

1987 ◽  
Vol 7 (12) ◽  
pp. 4204-4210
Author(s):  
M A Osley ◽  
D Lycan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fusion Gene ◽  
Chromosome Replication ◽  
Lacz Fusion ◽  
Histone Genes ◽  
Promoter Elements ◽  
Regulatory Mutations ◽  
Cycle Dependent ◽  
Beta Galactosidase

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

scholarly journals Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae

1998 ◽  
Vol 143 (1) ◽  
pp. 23-34 ◽  
Author(s):  
M. Oakes ◽  
J.P. Aris ◽  
J.S. Brockenbrough ◽  
H. Wai ◽  
L. Vu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Nuclear Periphery ◽  
Rdna Locus ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol I ◽  
Rdna Expression

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.

scholarly journals Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (9) ◽  
pp. 5408-5417 ◽  
Author(s):  
T Maeda ◽  
A Y Tsai ◽  
H Saito
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phosphatase Activity ◽  
Protein Tyrosine Phosphatase ◽  
Protein Phosphatase ◽  
Fusion Gene ◽  
Tyrosine Phosphatase ◽  
Growth Defect ◽  
Glutathione S Transferase ◽  
Protein Tyrosine ◽  
Colony Color

Two protein tyrosine phosphatase genes, PTP1 and PTP2, are known in Saccharomyces cerevisiae. However, the functions of these tyrosine phosphatases are unknown, because mutations in either or both phosphatase genes have no clear phenotypic effects. In this report, we demonstrate that although ptp2 has no obvious phenotype by itself, it has a profound effect on cell growth when combined with mutations in a novel protein phosphatase gene. Using a colony color sectoring assay, we isolated 25 mutants in which the expression of PTP1 or PTP2 is required for growth. Complementation tests of the mutants showed that they have a mutation in one of three genes. Cloning and sequence determination of one of these gene, PTC1, indicated that it encodes a homolog of the mammalian protein serine/threonine phosphatase 2C (PP2C). The amino acid sequence of the PTC1 product is approximately 35% identical to PP2C. Disruption of PTC1 indicated that the PTC1 function is nonessential. In contrast, ptc1 ptp2 double mutants showed a marked growth defect. To examine whether PTC1 encodes an active protein phosphatase, a glutathione S-transferase (GST)-PTC1 fusion gene was constructed and expressed in Escherichia coli. Purified GST-PTC1 fusion protein hydrolyzed a serine phosphorylated substrate in the presence of the divalent cation Mg2+ or Mn2+. GST-PTC1 also had weak (approximately 0.5% of its serine phosphatase activity) protein tyrosine phosphatase activity.

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides

2004 ◽  
Vol 45 (6) ◽  
pp. 371-377 ◽  
Author(s):  
Antonio Velayos ◽  
M�nica Fuentes-Vicente ◽  
Ra�l Aguilar-Elena ◽  
Arturo P. Eslava ◽  
Enrique A. Iturriaga
Keyword(s):  
Mucor Circinelloides ◽  
Prenyl Diphosphate ◽  
Prenyl Diphosphate Synthase

scholarly journals Heteromer formation of a long-chain prenyl diphosphate synthase from fission yeast Dps1 and budding yeast Coq1*

FEBS Journal ◽  
2008 ◽  
Vol 275 (14) ◽  
pp. 3653-3668 ◽  
Author(s):  
Mei Zhang ◽  
Jun Luo ◽  
Yuki Ogiyama ◽  
Ryoichi Saiki ◽  
Makoto Kawamukai
Keyword(s):  
Fission Yeast ◽  
Budding Yeast ◽  
Prenyl Diphosphate ◽  
Long Chain ◽  
Prenyl Diphosphate Synthase

scholarly journals Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (6) ◽  
pp. 3060-3069 ◽  
Author(s):  
S Irniger ◽  
C M Egli ◽  
G H Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Signal Sequence ◽  
Test System ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Elements ◽  
Polyadenylation Sites ◽  
Point Mutagenesis

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

scholarly journals Signals sufficient for 3'-end formation of yeast mRNA.

1996 ◽  
Vol 16 (6) ◽  
pp. 2772-2776 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Yeast Saccharomyces Cerevisiae ◽  
Synthetic Signal ◽  
A Site

The following three elements were previously shown to be required for 3'-end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA or related sequences, which function by enhancing the efficiency of downstream positioning elements; (ii) the positioning element AATAAA or related sequences, which position the poly(A) site; and (iii) the actual poly(A) site, which is usually Py(A)n. In this study, we synthesized a 39-pb poly(A) signal that contained the optimum sequences of these three elements. By inserting the synthetic 3'-end-forming signal into various positions of a CYC1-lacZ fusion gene, we showed that truncated transcripts of the expected sizes were generated. Furthermore, the poly(A) sites of the truncated transcripts were mapped to the expected poly(A) site within the synthetic signal. Our findings establish that the three elements are not only necessary but also sufficient for mRNA 3'-end formation in S. cerevisiae.

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