scholarly journals Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis.

1987 ◽  
Vol 7 (12) ◽  
pp. 4369-4376 ◽  
Author(s):  
J M Leonardo ◽  
S M Bhairi ◽  
R C Dickson
Keyword(s):  
Gene Expression ◽  
Deletion Mutant ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Base Sequence ◽  
Promoter Deletion ◽  
Deletion Mutations ◽  
Maximum Induction ◽  
Sequence Elements ◽  
Beta Galactosidase

Transcription of the Kluyveromyces lactis beta-galactosidase gene, LAC4, is inducible by galactose and lactose. We examined the effects of deletion mutations within the LAC4 promoter on the expression of beta-galactosidase activity. The results of these experiments indicate that at least two upstream activator sequences (UAS) mediate maximum induction by galactose. These UAS sequence elements are homologous to UAS that regulate induction of the melibiose-galactose regulon of Saccharomyces cerevisiae. We also show that a synthetic copy of one of the K. lactis UAS restores the inducibility of a deleted, noninducible LAC4 promoter. Since the uninduced or basal level of LAC4 expression was increased in several promoter deletion strains and in deletion strains carrying one or two synthetic UAS, we examined the contribution of the LAC9 positive regulatory protein to this effect. The LAC9 protein is thought to bind to UAS and activate transcription of LAC4 (L.V. Wray, M.M. Witte, R.C. Dickson, and M.I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987). Our results demonstrate that LAC9 protein plays a role in setting the uninduced level of gene expression, but other factors also participate. For example, in a lac9 background a LAC4 promoter deletion mutant with two copies of a synthetic 17-base-pair UAS yields a sevenfold higher level of uninduced LAC4 expression than the same strain with one UAS. These and other data indicate that the basal level of gene expression is strongly influenced by the base sequence of the promoter.

Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis

1987 ◽  
Vol 7 (12) ◽  
pp. 4369-4376
Author(s):  
J M Leonardo ◽  
S M Bhairi ◽  
R C Dickson
Keyword(s):  
Gene Expression ◽  
Deletion Mutant ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Base Sequence ◽  
Promoter Deletion ◽  
Deletion Mutations ◽  
Maximum Induction ◽  
Sequence Elements ◽  
Beta Galactosidase

Transcription of the Kluyveromyces lactis beta-galactosidase gene, LAC4, is inducible by galactose and lactose. We examined the effects of deletion mutations within the LAC4 promoter on the expression of beta-galactosidase activity. The results of these experiments indicate that at least two upstream activator sequences (UAS) mediate maximum induction by galactose. These UAS sequence elements are homologous to UAS that regulate induction of the melibiose-galactose regulon of Saccharomyces cerevisiae. We also show that a synthetic copy of one of the K. lactis UAS restores the inducibility of a deleted, noninducible LAC4 promoter. Since the uninduced or basal level of LAC4 expression was increased in several promoter deletion strains and in deletion strains carrying one or two synthetic UAS, we examined the contribution of the LAC9 positive regulatory protein to this effect. The LAC9 protein is thought to bind to UAS and activate transcription of LAC4 (L.V. Wray, M.M. Witte, R.C. Dickson, and M.I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987). Our results demonstrate that LAC9 protein plays a role in setting the uninduced level of gene expression, but other factors also participate. For example, in a lac9 background a LAC4 promoter deletion mutant with two copies of a synthetic 17-base-pair UAS yields a sevenfold higher level of uninduced LAC4 expression than the same strain with one UAS. These and other data indicate that the basal level of gene expression is strongly influenced by the base sequence of the promoter.

scholarly journals Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

1987 ◽  
Vol 7 (12) ◽  
pp. 4400-4406 ◽  
Author(s):  
K D Breunig ◽  
P Kuger
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Protein Binding Site ◽  
Regulatory Sequence ◽  
Functional Homology ◽  
Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

scholarly journals The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

1992 ◽  
Vol 12 (5) ◽  
pp. 1924-1931 ◽  
Author(s):  
N Kuzhandaivelu ◽  
W K Jones ◽  
A K Martin ◽  
R C Dickson
Keyword(s):  
Glucose Repression ◽  
Rotational Symmetry ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Genetic Regulation ◽  
Structural Genes ◽  
Important Principle ◽  
Biological Phenomena ◽  
Beta Galactosidase

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

scholarly journals Insight into the Kluyveromyces lactis Pdr1p regulon

2016 ◽  
Vol 62 (11) ◽  
pp. 918-931
Author(s):  
Nora Toth Hervay ◽  
Alexandra Konecna ◽  
Zuzana Balazfyova ◽  
Alexandra Svrbicka ◽  
Yvetta Gbelska
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Multidrug Resistance ◽  
Deletion Mutant ◽  
Dna Microarrays ◽  
Efflux Pump ◽  
Kluyveromyces Lactis ◽  
Binding Motifs ◽  
Almost All ◽  
Insight Into

The overexpression of efflux pumps is an important mechanism leading to the development of multidrug resistance phenomenon. The transcription factor KlPdr1p, belonging to the Zn2Cys6 family, is a central regulator of efflux pump expression in Kluyveromyces lactis. To better understand how KlPDR1-mediated drug resistance is achieved in K. lactis, we used DNA microarrays to identify genes whose expression was affected by deletion or overexpression of the KlPDR1 gene. Eighty-nine targets of the KlPDR1 were identified. From those the transcription of 16 genes was induced in the transformant overexpressing KlPDR1* and simultaneously repressed in the Klpdr1Δ deletion mutant. Almost all of these genes contain putative binding motifs for the AP-1-like transcription factors in their promoters. Furthermore, we studied the possible interplay between KlPdr1p and KlYap1p transcription factors. Our results show that KlYap1p does not significantly contribute to the regulation of KlPDR1 gene expression in the presence of azoles. However, KlPDR1 expression markedly increased in the presence of hydrogen peroxide and hinged upon the presence of KlYap1p. Our results show that although both KlPdr1p and KlYap1p transcription factors are involved in the control of K. lactis multidrug resistance, further studies will be needed to determine their interplay.

scholarly journals Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369 ◽  
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site

1987 ◽  
Vol 7 (12) ◽  
pp. 4400-4406
Author(s):  
K D Breunig ◽  
P Kuger
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Protein Binding Site ◽  
Regulatory Sequence ◽  
Functional Homology ◽  
Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene

1992 ◽  
Vol 12 (5) ◽  
pp. 1924-1931
Author(s):  
N Kuzhandaivelu ◽  
W K Jones ◽  
A K Martin ◽  
R C Dickson
Keyword(s):  
Glucose Repression ◽  
Rotational Symmetry ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Genetic Regulation ◽  
Structural Genes ◽  
Important Principle ◽  
Biological Phenomena ◽  
Beta Galactosidase

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

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