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L Guarente

Publications and source records attributed to L Guarente.

125 records · Page 7Linked to original sources
PubMed1985

Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae.

Transcription initiation of the yeast iso-1-cytochrome c gene (CYC-1) occurs in six major clusters at positions +1, +10, +16, +25, +34, and +43. Potential "TATA elements" lie upstream at positions -154, -106, -52, and -22. Analysis of the TATA region suggests that three of these TATA sequences are functional and contribute to initiation at CYC-1, with the -106 TATA promoting initiation at +1, +10, and +16; the -52 TATA, at +16, +25, +34, and +43; and the -22 TATA, at +34 and +43. Deletions changing the spacing between the TATA sequences and the region of transcription initiation do not change the location of the CYC-1 transcription start points. This finding suggests that at least part of the information determining mRNA initiation sites is encoded within the DNA sequence at the site of transcription initiation. Analysis of 18 yeast RNA polymerase II promoters suggests that two classes of DNA sequences serve as preferred sites for transcription initiation. To test this possibility, we replaced some of the normal CYC-1 start sites with one of these sequences, TCGA, and found that transcription initiates at this newly introduced sequence. These results are in contrast to those from higher eukaryotes, where RNA polymerase II typically initiates transcription a fixed distance downstream from the TATA element. The presence of multiple, functional TATA sequences at CYC-1 is inconsistent with the idea that RNA polymerase or another transcription factor attaches to the template at an upstream activation site and scans for the nearest TATA element.

Base Sequence↗
PubMed1985

Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae.

We report here the cloning of the HAP2 gene, a locus required for the expression of many cytochromes and respiratory functions in Saccharomyces cerevisiae. The cloned sequences were found to direct integration of a marked vector to the chromosomal HAP2 locus, and derivatives of these sequences were shown to yield chromosomal disruptions with a Hap2- phenotype. The gene maps 18 centimorgans centromere proximal to ade5 on the left arm of chromosome VII, distinguishing it from any other previously characterized nuclear petite locus. The HAP2 locus encodes a 1.3-kilobase transcript which is present at extremely low levels and which is derepressed in cells grown in media containing nonfermentable carbon sources. Levels of HAP2 mRNA are not reduced in strains bearing a mutation at the HAP3 locus, which is also required for expression of respiratory functions. Models outlining possible interactions of the products of the HAP2 and HAP3 genes are presented.

Carbohydrate Metabolism↗
PubMed1984

Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae.

The upstream activation site (UAS) of the yeast CYC1 gene is shown to contain two homologous subsites, UAS1 and UAS2. Each site, when placed upstream of the transcriptional initiation region of the yeast LEU2 gene, activates LEU2 transcription which is regulated by catabolite repression. UAS1 is responsible for most of the transcription under glucose repressed conditions, while UAS1 and UAS2 contribute equally to lactate derepressed transcription. A single point mutation in UAS2 increases its activity in glucose 10- to 20-fold. Several experiments indicate that UAS1 and UAS2 are regulated distinctly at the molecular level. First, UAS1 but not UAS2 is fully depressed in glucose by increasing the levels of intracellular heme. Second, trans-acting regulatory mutations, hap1-1 and hap2-1, selectively abolish the activity of UAS1 or UAS2. HAP1 appears to encode a protein that mediates catabolite repression of UAS1 by responding to intracellular heme levels.

Cytochrome c Group↗
PubMed1984

Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box".

The ability of the upstream activation sites (UASs) of the yeast CYC1 gene to function when inverted or when positioned downstream of the "TATA box" is investigated. Inversion of a 130-base-pair DNA fragment bearing the UASs leaves the activity of the sites almost completely intact. In contrast, positioning the sites downstream of the TATA box or in the intron of a CYC1-ribosomal protein 51-lacZ tribrid gene almost totally abolishes their activity. In the latter construct, the separation between the UASs and TATA box is roughly equivalent to that between the elements in the intact CYC1 promoter region. The UASs are shown not to interrupt transcription of splicing in this construct since a GAL10 UAS positioned upstream of the TATA box gives rise to galactose-inducible expression of the tribrid gene. The inability of the UASs to function in the intron is partly due to sequences between the intron and the TATA box that block the activation signal. However, a large component of the inactivity of the sites in the intron appears to be their downstream location. This result is discussed in light of possible mechanisms of upstream activation in yeast.

Base Composition↗
PubMed1983

Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site.

We show that expression of the iso-1-cytochrome c gene of Saccharomyces cerevisiae, CYC1, is tightly regulated by levels of intracellular heme. Expression is reduced at least 200-fold in cells grown under conditions of heme deficiency. Studies on the regulation of a CYC1-lacZ fused gene and direct determination of mRNA levels indicate that this control is transcriptional. Furthermore, we show that the heme regulatory site in the CYC1 promoter region is an upstream activation site (UASc) centered about 275 bp upstream from the region of transcriptional initiation. The latter region is required for optimal transcription and contains three TATA box sequences and six prominent mRNA initiation sites that span 34 bp. Substitution of the UASc with the UAS of the yeast GAL10 gene results in activation of the normal set of CYC1 transcripts. In this case, however, transcription is independent of regulation by heme, suggesting that in the wildtype, heme controls initiation per se and not translation or mRNA stability.

Cytochrome c Group↗
PubMed1983

In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli.

Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.

Alkaline Phosphatase↗
PubMed1982

Open reading frame cloning: identification, cloning, and expression of open reading frame DNA.

A plasmid was constructed that facilitates the cloning and expression of open reading frame DNA. A DNA fragment containing a bacterial promoter and the amino terminus of the cI gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacZ gene. An appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream of the lacZ-encoding DNA. This cloning vehicle produces a relatively low level of beta-galactosidase activity when introduced into Escherichia coli. The insertion of foreign DNA at the cloning site can reverse the frameshift mutation and generate plasmids that produce a relatively high level of beta-galactosidase activity. A large fraction of these plasmids produce a fusion protein that has a portion of the lambda cI protein at the amino terminus, the foreign protein segment in the middle, and the lacZ polypeptide at the carboxyl terminus. The production of a high level of beta-galactosidase and a large fusion polypeptide guarantees the cloning of a DNA fragment with at least one open reading frame that traverses the entirety of the fragment. Hence, the method can identify, clone, and express (as part of a larger fusion polypeptide) open reading frame DNA from among a large collection of DNA fragments.

Base Sequence↗
PubMed1982

A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site.

We have identified the promoter region of the GAL10 gene (whose product is UDP-galactose epimerase) of Saccharomyces cerevisiae; this promoter mediates galactose induction of transcription in conjunction with the product of the GAL4 regulatory gene. This identification was achieved by excising a 365-base-pair fragment of GAL10 leader DNA with a GAL10 proximal endpoint greater than 100 base pairs upstream of the transcriptional start site and substituting it in place of the upstream activation site of the CYC1 (iso-1-cytochrome c) promoter [Guarente, L. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2199-2203]. The hybrid promoter is composed of DNA encoding CYC1 mRNA start sites and the GAL segment upstream of these sites. This promoter is regulated in a manner analogous to GAL10; i.e., it is induced by galactose and responds to mutations in the GAL4 and GAL80 regulatory loci. The activity of the hybrid promoter requires sequences in the region of the CYC1 mRNA start sites but does not require a precise spacing between these sequences and the GAL segment. The transposed GAL segment appears not to contain sequences that mediate glucose repression. Thus, the picture of the GAL10 promoter that emerges is one of an upstream activation site that responds to the GAL4 product plus galactose, and a region of transcription initiation that may contain sequences that mediate glucose repression. Experiments employing strains inducible (GAL80) or constitutive (gal80) for GAL10 expression indicate that an additional component of glucose repression is inducer exclusion.

Base Sequence↗
PubMed1982

Mutant lambda phage repressor with a specific defect in its positive control function.

The lambda phage repressor is both a positive and a negative regulator of gene transcription. We describe a mutant lambda phage repressor that has specifically lost its activator function. The mutant binds to the lambda phage operator sites and represses the lambda phage promoters PR and PL. However, it fails to stimulate transcription from the promoter PRM. The mutation lies in that portion of repressor--namely, the amino-terminal domain--that has been shown [Sauer, R. T., Pabo, C. O., Meyer, B. J., Ptashne, M. & Backman, K. C. (1979) Nature (London) 279, 396-400] to mediate stimulation of PRM. We suggest that the mutation has altered that region of repressor which, in the wild-type, contacts RNA polymerase to activate transcription from PRM.

Bacteriophage lambda↗
PubMed1981

Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae.

Hybrid genes between the Escherichia coli lacZ gene and the iso-1-cytochrome c (CYC1) gene of Saccharomyces cerevisiae were constructed by recombination in vitro. Each of the hybrid genes encodes a chimeric protein with a cytochrome c moiety at the amino terminus and an active beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) moiety at the carboxy terminus. When these hybrids are introduced into S. cerevisiae on plasmid vectors, they direct synthesis of beta-galactosidase. beta-Galactosidase levels directed by one such plasmid display the pattern of regulation normally seen for cytochrome c (i.e., a reduction of synthesis in cells grown in glucose). This plasmid contains one codon of CYC1 fused to lacZ, and the fused gene is preceded by the 1100 nucleotides that lie upstream from CYC1. An analysis of deletions in the upstream DNA suggests that sequences required for efficient transcription initiation of CYC1 lie within the DNA segment 250--700 base pairs upstream from the start of the CYC1 coding sequence. This region is at least 130 base pairs upstream from the "Hogness box" sequence that precedes the CYC1 coding sequence.

Animals↗
PubMed1980

A technique for expressing eukaryotic genes in bacteria.

Methods are described that allow efficient expression in Escherichia coli of cloned eukaryotic genes. The methods require that the coding sequence of the gene in question be available in a form uninterrupted by intervening sequences (for example, as a complementary DNA clone). The gene products are synthesized unfused to other amino acid sequences. The genetic manipulations are simple, and require the plasmids described and commercially available enzymes.

Cloning, Molecular↗
PubMed1980

Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin.

In this paper we describe a method for constructing E. coli plasmids that direct efficient expression of genes that encode eucaryotic or procaryotic proteins. No functional assays for the proteins are needed, and they are produced in their native, unfused state. The only requirement is that the genes be isolable without intervening sequences. We describe as an example the construction of a plasmid that directs the synthesis of about 10,000-15,000 monomers per cell of rabbit beta-globin. The essential steps in a typical construction are as follows. --A region of the gene encoding the amino-terminal portion of the protein is fused to DNA encoding an enzymatically active carboxy terminal fragment of beta-galactosidase. The latter is carried on one of three plasmids designed to facilitate the fusion (the construction of these three plasmids is described in the Appendix). --A "portable promoter" of the lac operon is placed at many positions in front of the fused gene using nucleases in vitro. Those promoter placements that elicit efficient expression of the fused gene are identified by the beta-galactosidase activity that they express. (In the special case we describe, plasmids identified as directing efficient expression of beta-globin were found to bear "hybrid" ribosome binding sites consisting of the Shine-Dalgarno sequence carried on the promoter fragment and the ATG of the beta-globin gene.) --The gene of interest is reconstituted intact, with the portable promoter in place, by recombination in vitro or in vivo.

Animals↗
PubMed1980

Expression of the human fibroblast interferon gene in Escherichia coli.

We applied the method of Guarente et al. [Guarente, L., Lauer, G., Roberts, T.M. & Ptashne, M. (1980) Cell 20, 543-553] to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene. Two plasmids were recovered. One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF. Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF. This activity is lower than that expected from the differential rate of synthesis of the protein. We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.

Base Sequence↗
PubMed1979

Structure of the malB region in Escherichia coli K12. II. Genetic map of the malE,F,G operon.

Starting with a strain containing a malK-lacZ fusion, a series of lambda plaque-forming phages which carry varying amounts of the malE,F operon have been isolated. We have used these phages to construct a deletion map of the malE,F operon. The construction of this deletion map has led to the identification of a new gene, malG. The malG gene is located distal to malF. The malG gene product is a protein required for the active transport of maltose and maltodextrins.

Biological Transport↗