Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (3) ◽  
pp. 1111-1121
Author(s):  
L V Wray ◽  
M M Witte ◽  
R C Dickson ◽  
M I Riley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metal Binding ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Functional Relationships ◽  
Functional Implications

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.

scholarly journals Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (3) ◽  
pp. 1111-1121 ◽  
Author(s):  
L V Wray ◽  
M M Witte ◽  
R C Dickson ◽  
M I Riley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metal Binding ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Functional Relationships ◽  
Functional Implications

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.

Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon

1993 ◽  
Vol 13 (12) ◽  
pp. 7566-7576
Author(s):  
F T Zenke ◽  
W Zachariae ◽  
A Lunkes ◽  
K D Breunig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Constitutive Expression ◽  
Indirect Evidence ◽  
Transcriptional Activator ◽  
Negative Regulator ◽  
Gene Encoding ◽  
Fold Induction

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

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.

Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae

1983 ◽  
Vol 3 (3) ◽  
pp. 360-370
Author(s):  
C L Denis ◽  
E T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Site ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Site Mapping ◽  
Nonfermentable Carbon Source ◽  
Restriction Site Mapping

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.

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 Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.

1983 ◽  
Vol 3 (3) ◽  
pp. 360-370 ◽  
Author(s):  
C L Denis ◽  
E T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Site ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Site Mapping ◽  
Nonfermentable Carbon Source ◽  
Restriction Site Mapping

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.

scholarly journals Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

1993 ◽  
Vol 13 (12) ◽  
pp. 7566-7576 ◽  
Author(s):  
F T Zenke ◽  
W Zachariae ◽  
A Lunkes ◽  
K D Breunig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Constitutive Expression ◽  
Indirect Evidence ◽  
Transcriptional Activator ◽  
Negative Regulator ◽  
Gene Encoding ◽  
Fold Induction

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

scholarly journals Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 4999-5009
Author(s):  
D W Griggs ◽  
M Johnston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Glucose Repression ◽  
Transcriptional Start Site ◽  
Regulatory Gene ◽  
Distal Region ◽  
Deletion Mapping ◽  
Positive Elements ◽  
Gal4 Expression ◽  
Weak Expression

The GAL4 gene of Saccharomyces cerevisiae (encoding the activator of transcription of the GAL genes) is poorly expressed and is repressed during growth on glucose. To determine the basis for its weak expression and to identify DNA sequences recognized by proteins that activate transcription of a gene that itself encodes an activator of transcription, we have analyzed GAL4 promoter structure. We show that the GAL4 promoter is about 90-fold weaker than the strong GAL1 promoter and at least 7-fold weaker than the feeble URA3 promoter and that this low level of GAL4 expression is primarily due to a weak promoter. By deletion mapping, the GAL4 promoter can be divided into three functional regions. Two of these regions contain positive elements; a distal region termed the UASGAL4 (upstream activation sequence) contains redundant elements that increase promoter function, and a central region termed the UESGAL4 (upstream essential sequence) is essential for even basal levels of GAL4 expression. The third element, an upstream repression sequence, mediates glucose repression of GAL4 expression and is located between the UES and the transcriptional start site. The UASGAL4 is unusual because it is not interchangable with UAS elements in other yeast promoters; it does not function as a UAS element when inserted in a CYC1 promoter, and a normally strong UAS functions poorly in place of UASGAL4 in the GAL4 promoter. Similarly, the UES element of GAL4 does not function as a TATA element in a test promoter, and consensus TATA elements do not function in place of UES elements in the GAL4 promoter. These results suggest that GAL4 contains a weak TATA-less promoter and that the proteins regulating expression of this regulatory gene may be novel and context specific.

Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids

1987 ◽  
Vol 7 (3) ◽  
pp. 1233-1241
Author(s):  
M Irani ◽  
W E Taylor ◽  
E T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Tata Box ◽  
Upstream Region ◽  
Competition Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activation Sequence ◽  
Activation Sequence

Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region. This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box. Introducing excess copies of ADR1, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression. Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.

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