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L Guarente

Publications and source records attributed to L Guarente.

53 records · Page 3Linked to original sources
PubMed1987

Yeast HAP1 activator binds to two upstream activation sites of different sequence.

We show that the HAP1 protein binds in vitro to the upstream activation site (UAS) of the yeast CYC7 gene. Strikingly, this sequence bears no obvious similarity to the sequence bound by HAP1 at UAS1 of the CYC1 gene. The CYC1 and CYC7 sites compete for binding to HAP1 and have comparable affinities for the protein. The gross features of the interaction of HAP1 with the two sites are similar: multiple major and minor groove contacts, spanning 23 bp, on one helical face, with a back-side major groove contact toward one end. The precise positions of the contacts differ, however. A mutant form of HAP1, HAP1-18, abolishes the ability of the protein to bind to UAS1 but not CYC7 DNA. Possible mechanisms for how a single protein recognizes two sequences are discussed.

Base Sequence↗
PubMed1987

Subcloning.

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Amino Acid Sequence↗
PubMed1987

Constitutive expression of the yeast HEM1 gene is actually a composite of activation and repression.

We show that HEM1 (encoding 5-aminolevulinate synthase) expression, while constitutive under all steady-state growth conditions tested, is activated by the HAP2-HAP3 global activation system that controls expression of apocytochromes. This finding creates a paradox because apocytochrome activation by HAP2-HAP3 is highly regulated, subject to induction by heme, and subject to further derepression by a shift from glucose medium to one containing a nonfermentable carbon source. We clarify this issue by showing that HEM1 is subject to two additional layers of control that mask regulatory changes. First is a second activation system acting at a site close to the HAP2-HAP3 target sequence that keeps HEM1 turned on under conditions of heme deficiency. Second is a regulated negative control site downstream of the upstream activation site that counteracts derepression in medium containing a nonfermentable carbon source. Thus, transcription of the constitutive gene is actually a composite of opposing regulatory sites. This complex regulatory arrangement may exist to allow HEM1 to be coordinated transiently with apocytochromes for transition to respiratory growth. Conversely, it may reflect the alteration of HEM1 from a regulated to a constitutive gene over evolution.

5-Aminolevulinate Synthetase↗
PubMed1987

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation.

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

Chromosome Mapping↗
PubMed1986

Two or more copies of Drosophila heat shock consensus sequence serve to activate transcription in yeast.

A synthetic oligonucleotide bearing the Drosophila heat shock consensus sequence confers heat inducibility on a CYC1-lacZ gene in Saccharomyces cerevisiae. This sequence CTGGAATTTTCTAGA was inserted in place of the upstream activation sites of the CYC1 promoter adjacent to CYC1 TATA boxes. These constructs were transformed into yeast and found to be heat-inducible when two or more inserts were present. The level of inducibility seemed to increase with the number of inserted sequences: however, the orientations of these sequences relative to each other did not have much effect.

Animals↗
PubMed1986

The overproducing CYP1 and the underproducing hap1 mutations are alleles of the same gene which regulates in trans the expression of the structural genes encoding iso-cytochromes c.

The CYP1 gene has previously been identified as coding for a positive trans active factor that activates the expression of CYC1 and CYP3, which are the structural genes for isol1- and iso2-cytochrome c. Two phenotypically distinct classes of CYP1 mutations can be obtained indicating that CYC1 and CYP3 are differentially regulated by the product of CYP1. The HAP1 gene codes for a product which has previously been proved to be necessary for the expression of the heme dependent CYC1-UAS1 cis regulatory sequence. In this article, we show by complementation and recombination that CYP1 and HAP1 are the same gene, moreover we identify hap1-1 as an iso2-cytochrome c underproducer mutation of the CYP1 gene.

Alleles↗
PubMed1986

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Several site-directed mutagenesis regimens were used to generate single- and multiple-base substitutions in the upstream activation site UAS1 of the Saccharomyces cerevisiae CYC1 gene. Mutations resulting in large reductions in activity of the site lie in two distinct regions. Six single-base changes in a region A, between -288 and -285, all resulted in a 15-fold reduction in activity. Synthetic sites built up solely of multimers of the -289 to -285 sequence ACCGA behaved as carbon catabolite-sensitive UASs. In addition, substitution mutations in a second region, at nucleotides -266 and -265, virtually eliminated UAS1 activity. These mutations abolished the binding of a heme-dependent protein factor in vitro. Thus, UAS1 contains two essential regions both of which are required for its activity.

Base Sequence↗
PubMed1986

The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix.

delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed.

5-Aminolevulinate Synthetase↗
PubMed1986

Saccharomyces cerevisiae contains two functional citrate synthase genes.

The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

Chromosome Mapping↗
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

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↗
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

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↗