pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters

Hermann, Hannes; Häcker, Udo; Bandlow, Wolfhard; Magdolen, Viktor

doi:10.1016/0378-1119(92)90079-5

Cited by 29 publications

(23 citation statements)

References 15 publications

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“…The PGK1 promoter was amplified from pMA91 (Mellor et al, 1983) using primers A and B. The GCY1 terminator was amplified from pYLZ-2 (Hermann et al, 1992) using primers C and D. The fragments were mixed and fused by PCR using primers A and D. A ribosomal DNA sequence (Nieto et al, 1999) was amplified with primers 5rDNA and 3rDNA. RKI1, RPE1, TAL1 and TKL1 ORFs (Cherry et al, 1998) were amplified from S. cerevisiae CBS 8066 chromosomal DNA with the primers indicated in Table 1 and cut with BamHI on flanking sites introduced by the PCR primers.…”

Section: Primersmentioning

confidence: 99%

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Johansson¹,

Hahn‐Hägerdal²

2002

Yeast

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Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxPzeocin-loxP cassette using pCRE3. The 2m-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counterselection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.

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Section: Primersmentioning

confidence: 99%

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Johansson¹,

Hahn‐Hägerdal²

2002

Yeast

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“…Promoter Truncations and Expression Studies-Expression from the PFY1 promoter was studied in the genetic background of strain W303-1A (39) transformed with the low copy (CEN6-based) lacZ expression vector pYLZ6 (40). Promoter truncations were made by PCR using the 5Ј primers H4, H3, H2, H1, H0, and H6, each equipped with a 5Ј-flanking EcoRI restriction site in combination with the reverse primers R1 or R2, which contained a BamHI site for ligation to the lacZ reporter of pYLZ6.…”

Section: Methodsmentioning

confidence: 99%

“…3Ј-adjacent to the first nucleosome in the PFY1-coding region, a wide nuclease-sensitive linker coincided with the 3Ј-end of the intron. The downstream adjacent non-positioned two nucleosomes were bounded by two consecutive hypersensitive sites that lay in the bidirectional overlapping termination region of the two convergently transcribed genes PFY1 and GCY1 (51,52), the 3Ј-mRNA ends of which overlap in this region by 37 nucleotides (40).…”

Section: Effect Of the Mutation Of The Reb1p Site On Pfy1 Expression mentioning

confidence: 99%

Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

Angermayr

Oechsner

Bandlow

2003

Journal of Biological Chemistry

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“…The plasmid pLD was constructed by fusing a 926-bp fragment encoding the complete DIT1/DIT2 intergenic region including both AUG start codons and the first 3Ϫ4 codons (11 bp) in frame to the lacZ gene of the yeast centromeric vector pYLZ-6 [12]. To this end the fragment was amplified by PCR using the primers Dit1PCR (5′-AGGGCGGATCCCTAGTAAATGTCAT-3′) and Dit2PCR (5′-AACACGGATCCTTTAACAACTCCAT-3′) which introduced terminal BamHI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

Sporulation‐specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p

Bogengruber

Eichberger

Briza

et al. 1998

European Journal of Biochemistry

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Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation. Transcription of these two divergently arranged genes is controlled by a common 900-bp intergenic region. Random mutagenesis of this promoter and tests with appropriate reporter constructs identified an 11-bp cis-acting palindromic sequence, DIT repressor element (DRE), as a major negative regulatory site during vegetative growth. Repression is exerted by DRE in conjunction with a mid-sporulation element (MSE)-like sequence situated 26 bp away. These sequence elements are both contained within the 76-bp negative regulatory element (NRE) defined previously [Friesen H., Hepworth, S. R. & Segall, J. (1997) Mol. Cell. Biol. 17, 123Ϫ134]. The activated form of Rim101p, a transcriptional inducer of the early meiotic gene IME1, enhances expression from the DIT1 promoter both in vegetative and sporulating cells. Activation by Rim101p does not seem to involve binding of Rim101p at either of the two cis-acting sites described here, since reporter constructs with both elements or most of the NRE deleted could still be activated by the short form of Rim101p.Keywords : Saccharomyces cerevisiae; transcriptional regulation; sporulation; spore wall formation.During sporulation of Saccharomyces cerevisiae several major stages of gene activation, for instance early, middle, mid/ late and late, can be discerned. While the regulation of early sporulation genes has been intensively investigated [1,2], transcriptional control mechanisms of middle, mid/late and late genes are still largely unknown. Previous work on SPS4, a gene activated during mid-sporulation, revealed an upstream activating sequence (UAS) element which is responsible for the stagespecific expression of this gene [3]. This element (UAS SPS4 ) is sufficient to activate a CYC1-lacZ reporter gene construct during mid-sporulation. This activation is independent of UME6, which is essential for early gene expression, but requires IME2, an early meiotic gene. An identical sequence element, designated MSE (mid-sporulation element) was found to be essential for mid-sporulation-specific expression of SPR3, a sporulation-specific member of the yeast septin gene family, homologous to the genes encoding bud neck filaments [4]. It was also identified in the promoters of SPS18 and SPS19 [5], SMK1 [6] and SPR2 [7]. While SPS4 and SPR3 are up-regulated during sporulation, SPR3 is, in addition, actively repressed during vegetative growth [8].The genes DIT1 and DIT2, which are essential for the formation of the outer spore wall, are transcribed during mid/late-sporulation [9]. Expression of these divergently transcribed genes is controlled by a 900-bp intergenic region. Deletion analysis of this promoter led to the identification of a stretch of 76 bp which Correspondence to R. Schricker, Institut für Genetik und Allgemeine Biologie, Universität Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, AustriaFax: ϩ43 662 8044144.Abbreviations. DIT1 and DIT2, genes essential for dityrosine synthesis ; DRE, DIT repre...

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pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters

Cited by 29 publications

References 15 publications

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

Sporulation‐specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p

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