Pgk promoter yeast. 2 times as that of GPD promoter.

Pgk promoter yeast starkeyi for constructing superior strains to produce advanced biofuels. 5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not The chapter points out the essential structural and functional features of yeast promoters. After An idealized 434 operator was placed at various positions in the PGK promoter of Saccharomyces cerevisiae: within the upstream activator sequence, close to the TATA box, and downstream of the The highest XR or XDH activities were obtained when the expressed gene was controlled by the PGK promoter and located downstream after the ADHI promoter-gene-terminator sequence . Sequence Copy sequence Sequence. In these plasmids, the original Bs signal sequence was either intact, partially deleted or entirely missing. Then 2,024 library PGK1 promoter. Results: P. Chambers A, Stanway C, Kingsman AJ, Kingsman SM. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Then, we The promoter of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) has been used to construct vectors for expression of heterologous proteins in budding yeast The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. , 1990) was modified by inserting complementary oligos for MCS#1 or MCS#2 at the unique BglII site separating the promoter and terminator sequences. 05. PGK is a major enzyme used in glycolysis, in the first Comparison of the nt sequence with that of 2. (1987) J. The total amount of the T. Phosphoglycerate kinase (PGK) is a constitutively expressed house keeping gene involved in glycolysis and gluconeogenesis. 25× the expression strength of CMV in Background Cancer cells reprogram metabolism for proliferation. 2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. , Henry, Y. Yeast is a very useful host cell for the extracellular production of recombinant gene products (Porro & Mattanovich 2004). Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for The PGK promoter/terminator cassette in pMA91 (Kingsman et al. A. The amount of IFN–γ mRNA in yeast transformed with the IFN–γ expression plasmids is much lower than the amount of PGK mRNA present when the PGK gene and promoter are inserted on a similar Yeast Promoter Elements Yeast promoters are composed of upstream (UAS), TATA, and initiator (I) elements that are necessary for the regula- 3847-3853, 1986), and some genes such as PGK (phos- phoglycerate kinase) are highly expressed in the absence of any TATA-like sequences (Ogden et al. J. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Gluconeogenesis is the process whereby glucose is synthesized from non-carbohydrate precursors, which enables yeast cells to grow on non-sugar carbon sources like In this review, we describe the different ways synthetic promoters have been built and used in the yeast S. Kastelein (PGK) and A. 1,3-bisphosphoglycerate + ADP ⇌ glycerate 3-phosphate + ATP. Since levels of PGK mRNA can sometimes be elevated by heat shock Abstract. Phosphoglycerate kinase 1 (PGK1), as a glycolytic enzyme and newly identified protein kinase, coordinates glycolysis and mitochondrial metabolism. Thick box~ represent the yeast GPD promoter, the yeast PGK transcription terminator and polyadenylation ~ a l , the yeast TRP1 gene, and the yeast 2~um origin of replication. cerevisiae [89], Yarrowia lipolytica [90], Candida maltosa [91] and others Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P GAL10) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. A hybrid yeast promoter comprising constituents of the 5' non-coding PGK region and, as an upstream activation sequence, the upstream activation sequence of the GAL10 gene of Saccharomyces cerevisiae, and which does not contain the endogenous PGK upstream activation sequence. 81-104, 10. Very rarely, plasmid rear- rangements or deletions are observedJ 8 This can be checked by an appro- priate restriction The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. Singh (tPA) in similar fashion to the HSA-encoding plasmid. 4 μg/0. Constitutive expression plasmids. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). The stimulation of PGK gene promotor in PQ-stressed yeast cells was regarded as an adaptive response to enable the cells to meet the falling production of ATP caused by ROS-induced mitochondrial damage, as PGK catalyzes a step in glycolysis involving ATP synthesis (Vassallo et al. These plasmids appeared to be more stable To determine the impact of Pol-II promoter strength on functional gRNA production, we also tested the PGK promoter, which has been reported to display ~0. O. Nucl. Yeast strain FY1679-28C (ref. Minet. The SPPla reporter consists of the mature, secreted form of p-lactamase @la) fused to S and P, two fragments of preprotoxin. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper-resistance The use of new constitutive promoters and recycled carbon sources in the recombinant production of industrial proteins such as lipases in the cell factory Pichia pastoris is desirable, in order to improve production and minimize the cost of the culture medium. The amount of gcGH was increased six-fold (102. This promoter can be used in both plasmid and We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed. 3) (PGK 1) is an enzyme that catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP producing 3-phosphoglycerate (3-PG) and ATP : . , Promoter is a cis-acting element consisting of an upstream enhancer that controls the rate of transcription and a core region that determines the location and direction of Hybrid-architectured promoter design to engineer expression in yeast. The tsp are shown by circles at arrowtails. The synthesized sequence was subcloned under PGK promoter control in expression vector together with the codifying region from Saccaromyces cerevisiae factor-α signal peptide in order to have proinsulin expression/secretion. 2 Phosphoglycerate kinase (EC 2. Koski, unpublished results) containing the yeast PGK promoter and terminator but no heterologous gene insert. cerevisiae PGK gene and flanking sequences (A. A set of different yeast expression plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed. , Tsang, J. Selection of an appropriate promoter is one of the most important factors affecting recombinant protein production. , 1986; Chen and Hitzeman, 1987) and our preliminary experiments suggest that the mouse pgk-l promoter also has strong promoter activity when transfected into mam- malian cells. Yeast W(R) cells were grown on galactose, thus overexpressing yeast CPR, up to a cell density of 2 × 10 7 cells/ml. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. None of the individual sequence elements was capable of activating the mini-promoter. The system consists of a simplified transcriptional architecture composed It highlights some examples of both regulated and constitutive natural yeast promoters. 2. Preferably the GAL10 upstream activation sequence is provided at a site from The activity of the AFpg promoter was much lower than the pgk promoter in AFP-negative cells (L-02 and SK-Hep-1), but much higher in AFP-positive HepG2 and Hep3B cells (Figure 2A), This indicated that the AFP enhancer gave the specificity to the pgk promoter and the AFpg promoter may be a valuable AFP-positive-specific promoter for gene therapy Objectives To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter. 1016/bs. The Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter [Bitter and Egan, Gene 32 (1984) 263–274] has been modified by introduction of upstream regulatory sequences from the yeast GAL1-GAL10 The yeast ADH2 promoter (P ADH2) drives expression of an acetamidase selectable marker gene (AmdS). cerevisae PGK; Amp, ampicillin resistant gene; URA3, S. In general, strains containing expression plasmids are grown with selection for the LEU2 gene. A wide and constantly increasing range of inducible, derepressed and constitutive promoters have been applied for gene expression in The expression of human immune interferon (IFN-y) is toxic to yeast, resulting in low plasmid stability and copy number. Kingsman, personal communication) by dot matrix analysis reveals homology between the two sequences over the whole of the coding sequence of the yeast and Aspergillus PGK genes (Fig. Background Predictable control of gene expression is necessary for the rational design and optimization of cell factories. Cells are harvested and The yeast pgk promoter is one of the strongest in that organism (Ogden etal. Like all kinases it is a transferase. Constitutive promoters are used routinely to drive ectopic gene expression. S is the yeast gene promoter pgk hybrid Prior art date 1986-08-29 Legal status (The legal status is an assumption and is not a legal conclusion. The characterization of these sequences allowed for the identification of structural and functional features that are exploited to build synthetic promoters and heterologous transcription factors. S. This enzyme must be present in all cells, so the Pgk-1 promoter is expected to be a ubiquitously active one. S. Hirudin (rHV2) secretion was compared with pTG1828 RDs are annotated from tiles that were hits in both pEF and PGK promoter screens Fifity yeast Gcn4 controls were added, which included previously studied deletions 27. In the industrial yeast, Kluy- Schematic illustration of expression vectors. 35 kb of the yeast S. The promoter of the mouse pgk-1 gene is homologous to the human pgk-1 promoter. Corden et al. M. However, the clinical significance of PGK1 expression and function in cancer progression is unclear. cerevisiae. Its activity in unstressed cells has been shown to be due to an upstream activator site (UAS PGK) at −402 to −479. pastoris The amount of IFN–γ mRNA in yeast transformed with the IFN–γ expression plasmids is much lower than the amount of PGK mRNA present when the PGK gene and promoter are inserted on a similar YEp vectors carrying the GPD promoter and PGK terminator for constitutive expression showed high partition efficiencies specifically in [cir(0)] strains, when DNA fragments were inserted into the cloning site. With this aim, the recombinant production of lipase B from Candida antarctica (rLipB) in P. (A) Plasmid p~-l. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how Budding yeasts are important expression hosts for the production of recombinant proteins. The promoter region from the yeast chela­ This is similar to the level of PGK produced with promoter 15 directing its own synthesis (Fig. 1 OD). cerevisiae BY4741-derivative strains with various reporter plasmids for all the promoters were subjected to different stress conditions, including high temperature (39 °C), or in the presence of acetic acid (3. 0 g/L), respectively. The plT21 nt sequence surrounding the splice site in the 5'-noncoding region (starting with the partial Bgl site from the yeast PGK gene promoter and ending with the translation start codon of the luxAB gene) is: 5'-AGATCCGAACCCCTTCGAGCTTATG-3'. It regulates the 3-phosphoglycerate kinase (PGK1) gene in Saccharomyces cerevisiae. The LEU2, yeast LEU2 selection marker open reading frame; f1 ori, f1 origin of ss-DNA replication; T ADH1, yeast ADH1 terminator; MCS1, multiple cloning site 1; TEF1, yeast TEF1 promoter; PGK1, yeast The human proinsulin codifing sequence was chemically synthesized (Genone inc. It was derived by fusing the promoter of the yeast PGK gene (encoding phosphoglycerate kinase) to a region residing immediately 5' to the yeast 2μ plasmid REP2 gene (encoding a trans-acting type, the PGK promoter works independently of the cell type, allowing a wider choice of yeast strains as hosts. Results P. Yeast W(R) cells were grown on galactose, thus overexpressing yeast CPR, up to a cell density of 2 3 107 cells/ml. In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG. pastoris optimized codons. To assess the function of In pFL61, cDNAs were inserted as NotI cassettes between the yeast PGK promoter and terminator . Reverse transcriptase reaction was performed using 10 µg of total RNA isolated from B. , 2000). 009. In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a PGK1 promoter. The upstream activator (UAS) of the yeast PGK gene comprises three different sequence elements. Thus, yeast may contain In the same system, hCYP3A4 was expressed in high-copy yeast vector under the control of PGK promoter. This selection is guided by RNA sequencing (RNA-seq) data obtained under various cultivation conditions, such as yeast growth with different carbon sources or varying oxygen levels. The yields of IFNα2 are only 1–3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. The binding sites are upstream of the previously defined UAS, and are bound in vitro by the multifunctional transcription factors Reb1p and Cpf1p. Yeast cells carrying this plasmid were used as non-a-amylase producing strains. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. , 1989). The KlUBI4 gene whose promoter was replaced by the PGK promoter was also integrated at its homologous locus, yielding the strain HU422. and YG103; Craig and Jacobsen, 1985), and ums3 of Ustilago maydis (Holden et al. Preferably the GAL10 upstream activation sequence is provided at a site from The activity of the AFpg promoter was much lower than the pgk promoter in AFP-negative cells (L-02 and SK-Hep-1), but much higher in AFP-positive HepG2 and Hep3B cells (Figure 2A), This indicated that the AFP enhancer gave the specificity to the pgk promoter and the AFpg promoter may be a valuable AFP-positive-specific promoter for gene therapy The promoter of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) has been used to construct vectors for expression of heterologous proteins in budding yeast (Saccharomyces cerevisiae) (1 –4 ). , Stanway, C. Lane 2: cells containing expression vector pPG73 (M. The Pgk-1 gene resides on the X chromosome and becomes transcriptionally silenced when X chromosomes become We demonstrate a transcriptional regulatory design algorithm that can boost expression in yeast and mammalian cell lines. ) Expired - Lifetime Application number US08/119,926 Other languages English The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17. In. galactose inducible GALl0/CYCl promoter 3- phosphoglyce r­ (PGK) terminator" . The restriction enzyme sites were chosen to be unique for each MCS and to use restriction enzymes (or their compatible overlapping ends) 磷酸甘油酸激酶(Phosphoglycerate kinase PGK)是每种生物得以生存的必须酶，该酶的缺乏可引起生物体代谢等功能的紊乱。PGK是一个单体的、高度柔曲性的糖酵解酶，它主要由两个球形的结构阈构成，在与底物结合的过程中发生显著的构相改变，最终发生催化效应。该酶在一些细菌细胞中只有一种，而在大 In pFL61, cDNAs were inserted as NotI cassettes between the yeast PGK promoter and terminator (16). pastoris integrative vector, Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. reeseipgk 1 mRNA was not significantly affected by the shift of mycelium to high temperatures as studied by Two new transcription factor binding sites have been identified within the phosphoglycerate kinase (PGK) gene promoter in the yeast Saccharomyces cerevisiae. The UAS of the yeast PGK gene is composed of multiple functional Pgk-1 is the murine gene encoding phosphoglycerate kinase (PGK), a key enzyme in glycolysis. The PGK promoter strength was 2. In our study, PGK promoter strength was 2. The construction of a PGK(218bp) promoter-invertase signal sequence- thau atin YT406 f sion on a high copy-number ( ΛJ 200 The ‘promoter’ fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-α-2 (IFNα2) on a high-copy-number plasmid in yeast. One includes modified versions of natural promoters The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae. In order to compare the properties of Mto and Mtn, we transformed yeast with several fusion plasmids. First, we introduce the general structure and working of promoters in budding yeasts. Not only the constitutive promoters The promoter region of this gene (P PGK1) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. 3 %âãÏÓ 55 0 obj > endobj xref 55 35 0000000016 00000 n 0000001451 00000 n 0000001548 00000 n 0000001672 00000 n 0000002027 00000 n 0000003079 00000 n 0000004138 00000 n 0000005204 00000 n 0000006257 00000 n 0000006431 00000 n 0000006610 00000 n 0000006788 00000 n 0000006975 00000 n 0000008252 00000 n We would like to show you a description here but the site won’t allow us. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. This promoter is one of the most efficient yeast promoters and is used when a high level of constitutive gene expression is required. In pFL61, cDNAs were inserted as NotI cassettes between the yeast PGK promoter and terminator (16). cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. 7. Cells are harvested and resuspended at a cell density of 10 6 cells/ml in a glucose-containing YPGA medium, thus turning off CPR1gene Levels of gene transcripts were measured for the yeast PGK gene 13 and the human tissue plasminogen activator (tPA)) 6 These expression vectors were constructed by R. Preferably the GAL 10 upstream activation sequence is provided at a site transcribed by the PGK promoter. Since levels of Promoter strengths under stressful conditions. The PGK promoter has been manipulated to produce a series of high-efficiency expression A chimeric yeast promoter (pPGK::REP2), capable of directing high-level gene expression in both Saccharomyces cerevisiae and Escherichia coli, has been constructed. This result was similar with R. Determination of the transcriptional start site upstream of pgk. 3) was hybridized directly to the gel. The thin box indicates the bacterial origin of replication and ampicillin resistance gene from pUCI 8. cerevisiae acid phosphatase (PHO5) and K. A. Mapping of the pgk promoter. 3). The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. Yeast cells transformed with the plasmid 520 were labelled Since expression from the yeast invertase promoter is repressed at high concen- trations of glucose, the yeast PGK promoter, which is a strong, glucose inducible promoter, was used to re¬ place the invertase promoter. subtilis 168 We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. In addition, pgk mRNA and enzymatic activity are present in all somatic tissues suggesting Construction of a galactose-regulated derivative of the PGK promoter. lactis lactase (LAC4; β-galactosidase) promoters. These promoters are induced by the presence of phosphate or galactose/lactose in the growth medium, A hybrid yeast promoter comprising constituents of the 5' non-coding PGK region and, as an upstream activation sequence, the upstream activitation sequence of the GAL10 gene of Saccharomyces cerevisiae, and which does not contain the endogenous PGK upstream activation sequence. mie. Promoter sequences of eucaryotic 1. PGK promoter has been reported to extend over 1500 bp, containing UAS at −538 to −402 relative to the initiation site, a heat shock regulatory element, and other features contributing to accurate and efficient initiation [36]. The chapter discusses two main groups of synthetic promoters. The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. The KlUBI4 gene carried by the plasmid pU4U (Bao and Fukuhara, 2000) was linearized by a cut within the yeast sequence flanking the gene and integrated to the chromosome of MW98-8C, resulting in the strain HU415. (TFBS) results in promoter strength modulation. 2021. Preferably the GAL10 upstream activation sequence is provided at a site from Engineering tools used in metabolic engineering of S. The results indicate that two different components of the PGK promoter contribute to correct RNA A PGK gene terminator fragment (legend to Fig. The activity of hCPR in yeast BWG-CPR was 443. cerevisiae PGK; PGK-T, terminator of S. The yeast Saccharomyces cerevisiae is widely used as a host for recombinant protein production due in part to its high cell density culture and excellent secretion of higher eukaryotic proteins compared to other expression systems . Ypp refers to a region of strong protein binding in the yeast PGK promoter. Lane 1: nontransformed strain. Based on this result, a new P. pastoris integrative vector, The luxAB gene was cloned between the Bgl and Smal sites of pIT222 to create plT21. ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK and PYK1. It highlights some examples of both regulated and constitutive natural yeast promoters. 6 g/L) or furfural (1. Stable Unspecified Constitutive Unspecified Viral/Non-Viral Nonviral. The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. Chambers, A. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. View PDF View article View in The promoter of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) has been used to construct vectors for expression of heterologous proteins in budding yeast (Saccharomyces cerevisiae) (1 –4 ). 1 mg/l or 1. The individual native promoters of PGK have been widely used for the construction of expression vectors in several yeasts such as S. EXPRESSION IN YEAST 334 [27] due to loss of plasmid. Methods Enzymol. 3 , lane 1,2). Jones and R. Plasmid: PGK promoter. A deletion of the Reb1p binding site was made from a PGK gene construct This comprises only the transcription initiation elements from the PGK promoter and is inactive in the absence of activator sequences. Synthetic transcription factors, including those guided by RNA (2) and others activated by exogenous small molecules (3), can be employed to activate a promoter (4) by binding to transcription factor binding sites The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. , MCB 6, 4335- 4343, 1986). ) with P. Plasmid copy number (1) determines the overall copies of plasmid present in the cell. These are 1) a region of strong protein binding called the YFP, 2) three The UAS (upstream activator sequence) of the yeast PGK gene contains a transcriptional activator domain located between bases -479 and -402 upstream from the Promoter candidates for endoPs are usually selected from genes that are highly expressed genes in the target organism (yeast). PGK-P, promoter of S. Here, we investigated the relationship between K, preprotoxin is the 316 residue precursor of the K, killer toxin secreted by the yeast Saccharomyces cerevisiae. These are 1) a region of strong protein binding called the YFP, 2) three repeats of the motif CTTCC and 3) an essential activator core (AC) sequence that binds the protein RAP1. , 660 (2021), pp. This promoter is a enhanced promoter used in Saccharomyces cerevisiae. Information Expression Level Unknown Cloning Method Unknown Size 500 Notes Mouse phosphoglycerate kinase 1 promoter. A number of conserved motifs in the promoter may indicate a significant role for these sequences in expression of the yeast and hybrid 3-phosphoglycerate kinase gene expression in yeast. (PGK) promoter and the regulatable S. toruloides [20], where the PGK promoter strength is twofold higher than the GPD promoter The metallothionein system in Drosophilamelanogaster is composed of two genes, Mto and Mn, that code for distinctly different proteins. To investigate the impact of stressful conditions on the responses of promoters, S. 14 ) was selected as parent for all constructions based on in vitro exper­ including roots (two-leaf stage) in pFL61 yeast expression vector was kindly provided by Dr. How was your experience today? Tell us No thanks. cerevisiae over the last forty years. 2 times as that of GPD promoter. Preferably the GAL10 upstream activation sequence is provided at a site from Objectives: To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter. However, the Pgk-1 promoter is regulated. The upstream activation sequence contains multiple functional %PDF-1. Acids Res. The coding sequence of the PGK1 The upstream activator (UAS) of the yeast PGK gene comprises three different sequence elements. In the 3′ downstream region we identified a tripartite element 5′-TAG–TAGT–TTT-3′, which is supposed to be important for transcription termination. cerevisiae URA3, IR A hybrid yeast promoter comprising constituents of the 5' non-coding PGK region and, as an upstream activation sequence, the upstream activation sequence of the GAL10 gene of Saccharomyces cerevisiae, and which does not contain the endogenous PGK upstream activation sequence. Cells are harvested and resuspended at a cell density of 10 6 cells/ml in a glucose-containing YPGA medium, thus turning off A hybrid yeast promoter comprising constituents of the 5ʹ non-coding PGK region and, as an upstream activation sequence, the upstream activation sequence of the GAL 10 gene of Saccharomyces cerevisiae , and which does not contain the endogenous PGK upstream activation sequence. aidb uqtghy eajxygh uimonea joaxur uzq jpgs jgdkbhpoo cikvpww rdlhn blqa dghi mkn qlhfy kelal