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K Struhl

Publications and source records attributed to K Struhl.

At least 145 records · Page 8Linked to original sources
PubMed1984

Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence.

DNA molecules created by fusing a 365-base-pair segment of yeast DNA encoding the galactose-regulated upstream promoter element (gal) to a set of derivatives that systematically delete sequences upstream from the his3 gene are introduced in single copy back into the yeast genome precisely at the his3 locus and then assayed for transcription. Fusions of the gal regulatory element to his3 derivatives containing all normal mRNA coding sequences but lacking essentially the entire promoter region fail to express his3 under any growth conditions. Fusions to derivatives lacking the his3 upstream promoter element but containing the "TATA box" place his3 expression under gal control--i.e., extremely high RNA levels in galactose-containing medium and essentially no his3 RNA in glucose-containing medium. However, of the two normal his3 initiation sites, only the downstream one is activated by the gal element. In fusions of this type, neither the orientation of the gal element nor the distance between the element and the his3 TATA box affects the level or the initiation points of transcription. However, the gal element does not influence transcription when placed 100 or 300 base pairs downstream from the normal mRNA start sites. Fusions to derivatives containing the entire his3 promoter region restore the basal level of his3 transcription in glucose-grown cells, and both transcriptional initiation sites are used. Furthermore, RNA levels in galactose-grown cells, although somewhat higher than in glucose-grown cells, are significantly below the fully induced level. The distance from his3 coding sequences does not affect RNA levels, suggesting that specific sequences, possibly corresponding to the his3 upstream promoter element, reduce the ability of the gal element to activate transcription. Analysis of chromatin from some of these strains indicates a DNase I-hypersensitive site(s) in the middle of the gal element. However, this structural feature is not correlated with transcriptional initiation because it is found when cells are grown in glucose medium and also in derivatives lacking a TATA box. Thus, the gal upstream element possesses most, but not all, of the properties of viral and cellular enhancer sequences of higher eukaryotes. In addition, it appears that the his3 and gal upstream sequences represent two distinct classes of promoter elements, which activate transcription from different initiation sites.

Alleles↗
PubMed1983

Direct selection for gene replacement events in yeast.

A method that facilitates gene replacement at the HIS3 locus of Saccharomyces cerevisiae (yeast) has been developed. First, an internal region of the cloned HIS3 gene was replaced by a DNA segment containing the wild-type ribosomal protein gene, CYH2. Second, by using standard yeast transformation methods, the wild-type HIS3 locus of a cycloheximide resistant strain (cyh2r) was replaced by this his3-CYH2 substitution. The resulting strain is sensitive to cycloheximide because CYH2 is dominant to cyh2r. Third, his3 mutations cloned into integrating or replicating vectors were introduced into this strain by selecting transformants via the vector-encoded marker. Selection for cycloheximide-resistant colonies resulted in the replacement of the his3-CYH2 allele by newly introduced his3 alleles. Thus, this scheme provides for the direct selection of gene replacement events at the HIS3 locus independently of the phenotype of the cloned his3 derivatives. In principle, it can be extended to any region of the yeast genome.

Alleles↗
PubMed1982

The yeast his3 promoter contains at least two distinct elements.

Phenotypic analysis of 65 mutations indicates that the yeast his3 promoter is composed of at least two separate regions of DNA. Each is necessary, but neither is sufficient for wild-type levels of his3 expression. Deletion mutations that destroy either promoter element express his3 poorly or not at all. The upstream element is located between 112 and 155 base pairs before the site of transcriptional initiation (nucleotides -112 to -155). A comparison of derivatives strongly suggests that the downstream element maps somewhere between nucleotides -32 and -52 and includes a sequence between nucleotides -45 and -52. This location coincides with sequences conserved before most eukaryotic genes(the TATA box region). By using derivatives in which his3 sequences are replaced by a small fragment of coliphage M13 DNA, three properties of the his3 promoter were established. First, his3 TATA box deletions fail to express his3 because they lack specific sequences and not because they disrupt spacing relationships between other sequences. Second, the TATA box region can be replaced functionally by the one orientation of the M13 DNA fragment that contains a TATA-like sequence. Third, the distance between the two elements (normally 90 base pairs) can be varied between 40 and 160 base pairs without markedly affecting promoter function. These results strongly suggest that yeast RNA polymerase II, unlike its Escherichia coli counterpart, does not bind simultaneously to both promoter elements, and they add further support to the view that the upstream element is not part of a transcriptionally competent binding site. This ability of the his3 upstream promotor element to act at a long and variable distance is similar to properties of viral enhancer sequences and is reminiscent of position effects in yeast.

Base Sequence↗
PubMed1981

Deletion mapping a eukaryotic promoter.

The phenotypes of 24 mutants that successively delete DNA sequences adjacent to the 5' end of the Saccharomyces cerevisiae (yeast) his3 structural gene are described. Deletions retaining greater than 155 base pairs before the mRNA coding sequences are phenotypically indistinguishable from the wild-type his3 allele. Deletions having end points between 113 and 65 base pairs before the transcription initiation site express his3 at reduced levels. Mutations retaining less than 45 base pairs are indistinguishable from null alleles of the his3 locus. These results indicate (i) that a sequence(s) located 113--155 base pairs upstream from the transcribed region is necessary for wild-type expression and (ii) that the T-A-T-A box (a sequence in front of most eukaryotic genes) is not sufficient for wild-type promoter function. Thus, the yeast his3 promoter region appears large when compared with prokaryotic promoters, suggesting that it may be more complex than a simple site of interaction between RNA polymerase and DNA.

Chromosome Deletion↗
PubMed1979

Isolation and characterisation of a yeast chromosomal replicator.

A yeast DNA sequence that behaves as a chromosomal replicator, ars1 (autonomously replicating sequence), has been isolated. On transformation, ars1 allows autonomous replication of all co-linear DNA. The replicator can integrate into other replication units and can function in multimeric form. The 850-base pair ars1 element has no detectable homology to other yeast sequences. Such replicator-containing plasmids can be used for the isolation of DNA sequences in yeast cells as well as for the study of chromosomal DNA replication.

Base Sequence↗
PubMed1979

Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments.

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.

Containment of Biohazards↗
PubMed1979

High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

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.

DNA Replication↗