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C F Wright

Publications and source records attributed to C F Wright.

28 records · Page 2Linked to original sources
PubMed1987

Structural and functional studies of the amino terminus of yeast metallothionein.

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.

Amino Acid Sequence↗
PubMed1987

Function and regulation of yeast copperthionein.

A functional copperthionein (CUP1) gene in Saccharomyces cerevisiae is essential to prevent copper-mediated cytotoxicity, but is dispensable for cell growth in the absence of exogenous copper. The CUP1 gene is negatively autoregulated, as observed by the necessity for a functional CUP1 gene in order to repress basal level transcription from the CUP1 promoter. Both the copper protection and transcriptional autoregulatory functions can be complemented by expression in yeast of either of two monkey metallothionein isoform cDNAs. The expression of the CUP1 gene is induced at the level of transcription by copper via cis-dominant upstream control sequences which are tandemly repeated. Synthetic CUP1 upstream control sequences confer copper inducibility on a heterologous yeast promoter in a manner similar to that observed for the authentic upstream control region.

Animals↗
PubMed1987

In vitro synthesis of vaccinia virus late mRNA containing a 5' poly(A) leader sequence.

We have shown that an extract made from HeLa cells harvested 6 hr after infection with vaccinia virus can transcribe a duplex DNA template containing a late viral gene. S1 nuclease analyses using genomic and synthetic probes indicated that the 5' ends of RNA synthesized in vitro are similar to those of RNA made in vivo and contain 5' poly(A) sequences contiguous with the translation initiation codon. Kinetic analysis of RNA synthesized in vitro demonstrated that a correctly initiated and 5' polyadenylylated product appeared within 5 min after transcription reactions were started. A cis-splicing mechanism of poly(A) addition can be ruled out because the DNA template used in vitro had no poly(dT) sequence and could contain as few as 37 base pairs upstream of the start of the RNA. In addition, we found that a point mutation in the first of two consecutively encoded adenylate residues preceding the ATG initiation codon abolished transcription in vitro. These data are consistent with at least three models: (i) RNA polymerase initiates RNA synthesis with a run of adenylate residues; (ii) a poly(A) primer is used for initiation; or (iii) the poly(A) leader is rapidly and efficiently attached to the RNA by ligation.

Cell-Free System↗
PubMed1986

Chromogenic identification of oligonucleotide-directed mutants.

We describe a simple plaque color assay for identifying oligonucleotide-directed mutations in cloned DNA fragments. The basis of the method is to: fuse the sequence of interest in-frame to the E.coli lacZ gene to produce a blue plaque phage, mutate the site of interest to a stop codon to generate a white plaque phage, and revert the stop codon and surrounding nucleotides to give a blue plaque phage containing one or more desired amino acid changes. The advantages of this cyclic method are that it produces truncated as well as amino acid substituted protein molecules, it can be repeated to introduce additional mutations, and it eliminates the need for labor intensive screening. Essentially any piece of DNA can be mutated using this method if the fragment has one open reading frame. If there is an open reading frame between the site and the lacZ gene, ATG codons can be inserted at the target site. We have used this method to produce termination and amino acid substitution mutants in the yeast CUP1 gene.

Base Sequence↗
PubMed1985

Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae.

The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base-pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.

Base Sequence↗
PubMed1984

A positive regulatory site and a negative regulatory site control the expression of the Saccharomyces cerevisiae CYC7 gene.

A series of BAL31 deletions were constructed in vitro in the upstream region of the Saccharomyces cerevisiae CYC7 gene, encoding the iso-2-cytochrome c protein. These deletions identified two sites which play a role in governing the expression of this gene. A positive site, the deletion of which led to decreased CYC7 expression, lay ca. 240 base pairs 5' to the translational initiation codon (-240). A negative site, the deletion of which led to greatly increased levels of CYC7 expression, lay at ca. -300 bp. Deletion of both these sites resulted in low wild-type-like expression of the gene. Therefore, these two sites appear to act antagonistically to give the low wild-type levels of CYC7 expression. Within the region defined as containing the positive site, there is a sequence which bears some homology to the upstream activation sites in the regulated gene, CYC1, encoding the iso-1-cytochrome c protein.

Base Sequence↗
PubMed1983

Expression of the yeast CYC genes and CYC1/GalK fusion genes on yeast plasmids.

We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids. The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed [such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid] and selected [as in the case of the IS1 insertion into the YCpCYC7(2) plasmid], and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes. Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid. Although we cannot rule out the possibility that use of plasmids might cause us to miss a class of regulatory effects that can be propagated only along a chromosomal structure, we believe that the regulatory effects that we do observe can be more quickly and completely defined by working with plasmids. If any regulatory effects occur only on chromosomes, they can be studied more easily once the basic regulatory phenomena have been analyzed. The regulatory regions of the CYC1, CYC7, and TR2 genes that we have crudely mapped so far all exert their effects 100-300 bp away from the putative transcriptional starting sites. How the information in these regions is transmitted along the DNA is an intriguing question. We are engaged in a mutational analysis of these sites to locate them more precisely, to map second-site mutations that moderate the effects of the original mutations, to obtain genomic mutations that define the genes whose products interact with these sites, and to test combinations of genomic and plasmid mutations to define the sites with which regulatory elements interact. This approach should aid our understanding of the spatial relationships between yeast regulatory sites and transcriptional signals. Ultimately, obtaining mutations in regulatory genes, such as the mutations described here for the anaerobic regulation of TR2, will allow the cloning of these genes by complementation. This will lead to the isolation of the protein encoded and ultimately to an approach to the molecular mechanism of regulation through study of protein-DNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Inducible repair of thymine ring saturation damage in phi X174 DNA.

The susceptibility to inducible SOS repair of 5,6-dihydroxy-dihydrothymine (t') damage in single-stranded phi X174 DNA has been measured. Following exposure to osmium tetroxide, which introduces t' damage in DNA under the conditions used, biological survival of the DNA infected into spheroplasts of Escherichia coli which had received prior exposure to ultraviolet light was higher than in unexposed spheroplasts. From our results, we conclude that approximately 63% of the biological damage from t' products, which is one of the classes of damage present in DNA following ionizing radiation, is susceptible to repair by the inducible SOS repair system.

Bacteriophage phi X 174↗