Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon.
01 Nov 1982-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 79, Iss: 22, pp 6971-6975
TL;DR: Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium, indicating that the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene GAL80.
Abstract: GAL4 is a classically defined positive regulatory gene controlling the five inducible structural genes of galactose/melibiose utilization in yeast. The positive regulatory function of the GAL4 gene product in turn is controlled by the product of another gene, the negative regulator GAL80. We have cloned a 3.1-kilobase fragment containing GAL4 by homologous complementation using the multicopy chimeric vector YEp24 and demonstrated that multiple copies of GAL4 in yeast have pronounced dosage effects on the expression of the structural genes. Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium. Multiple copies of the GAL4 plasmid also increase expression of the structural genes in inducing (galactose) medium and can partially overcome the effects of a dominant super-repressor mutant, GAL80S. Using an internal deletion in GAL4, we have demonstrated that these dosage effects are due to overproduction of GAL4 positive regulatory product rather than an effect of the flanking sequences titrating out a negative regulator. These results point to the importance of competitive interplay between the positive and negative regulatory proteins in the control of this system. We have also used the dosage effect of GAL4 plasmid in combination with different GAL4 and GAL80 alleles to create new phenotypes. We interpret these phenotypes as indicating that (i) the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene, GAL80, and (ii) the GAL80 protein may have specific interactions with the control regions of the structural genes.
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TL;DR: Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmID sequences.
1,641 citations
TL;DR: The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication in Drosophila melanogaster.
Abstract: A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.
1,033 citations
Cites background from "Isolation of the yeast regulatory g..."
...Another strategy for refining expression patterns in GAL4 lines is targeted suppression of GAL4 activity in a subset of cells comprising the pattern by expression of GAL80 (Lee and Luo 1999); GAL80 binds to GAL4 and neutralizes its transcription-promoting activity (Yun et al. 1991; Traven et al.…...
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...The successful implementation of this strategy requires that GAL80 be expressed at a sufficiently high level ( Johnston and Hopper 1982; Salmeron et al. 1989)....
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TL;DR: Two short regions of GAL4 are identified, each of which activates transcription when fused to the DNA-binding region of the molecule, which is not required for gene activation.
834 citations
Cites background from "Isolation of the yeast regulatory g..."
...Introduction GAL4, a protein of 881 amino acids, is a transcriptional activator of genes required for galactose catabolism in the yeast S. cerevisiae (Oshima, 1982; Johnston and Hopper, 1982; Laughon and Gesteland, 1982, 1984; Hashimoto et al., 1983)....
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TL;DR: It is shown that the yeast positive regulatory protein GAL4 binds to four sites in the upstream activating sequence UASG to activate transcription of the adjacent GAL1 and GAL10 genes, consistent with the idea that GAL2 protein binds to three related 17 bp sequences, each of which displays approximate 2-fold rotational symmetry.
694 citations
681 citations
References
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TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
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TL;DR: A simple and rapid method for preparing plasmids for restriction enzyme analysis has been developed and can be readily adapted for the preparation of plasmid from liter cultures with quantitative yields.
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TL;DR: This work has used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced.
Abstract: A stable leu2- yeast strain has been transformed to LEU2+ by using a chimeric ColE1 plasmid carrying the yeast leu2 gene. We have used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced. These studies show that ColE1 DNA together with the yeast sequences can integrate into the yeast chromosomes. This integration may be additive or substitutive. The bacterial plasmid sequences, once integrated, behave as a simple Mendelian element. In addition, we have determined the genetic linkage relationships for each newly introduced LEU2+ allele with the original leu2- allele. These studies show that the transforming squences integrate not only in the leu2 region but also in several other chromosomal locations.
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TL;DR: A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized in this paper.
Abstract: A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.
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