Biomedical subjects

R S Zitomer

Publications and source records attributed to R S Zitomer.

At least 37 records · Page 2Linked to original sources

PubMed1991

The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.

Alleles↗

PubMed1990

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels.

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.

Blotting, Southern↗

PubMed1990

A sequence that directs transcriptional initiation in yeast.

While RNA polymerase II of the yeast Saccharomyces cerevisiae initiates transcription at discrete sites, these sites are located over a wide range of distances from the TATA box for different genes. This variability has led to a number of proposals for consensus sequences located at the initiation site which, in conjunction with the TATA box, would direct initiation. We tested this hypothesis via oligonucleotide-directed mutagenesis, by placing the sequence CAAG, a member of one of these consensus sequences, upstream of the coding sequence of the CYC7 gene at a site at which initiation does not occur. The distance between the TATA sequence and this putative initiation site was varied by inserting it into the wild-type gene and three deletion mutants. The results demonstrated that this sequence can serve as an initiation site when located 49, 77, or 106 bp from the TATA sequence, but not when located 30 bp away.

Base Sequence↗

PubMed1990

A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae.

We have identified a consensus operator sequence, YYYATTGTTCTC, which mediates the repression imposed by the ROX1 factor upon the members of the hypoxic gene regulon, which includes ANB1, HEM13, COX5b, and CYC7. The members of the regulon were repressed with widely varying stringency, and the variation was correlated with the number and fidelity of operator sequences observed. ANB1 had two operators operating with unequal efficiency, each containing two copies of the operator sequence. Synthetic operator sequences introduced into an operator deletion were effective as monomers but much more so as dimers, consistent with cooperativity. The native operators both imposed ROX1 repression on the GAL1 gene, in either orientation, but the synthetic operators did not, indicating that the sequence context may be important. The repression and activation of ANB1 are independent spatially and functionally, since deletion of the operators did not reduce expression and since both the operator and activation regions functioned separately in the GAL1 UAS. The ANB1 UAS was constitutive, containing several elements distributed over a 300-bp region. There were two dT-rich segments, one of 51 bp and one of 165 bp, the latter capable of activating transcription by itself. Flanking segments containing GRF2 (REB1) and ABF1 (GF1) sites may contribute to activation but were not essential. The UAS showed a strongly preferred orientation.

Base Sequence↗

PubMed1989

Regulated overproduction of alpha-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis.

High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.

Blotting, Southern↗

PubMed1988

ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae.

The ROX1 gene encodes a product implicated in the regulation of heme-repressed and heme-induced genes in Saccharomyces cerevisiae. The gene has been cloned and shown to code for a 1.4-kilobase transcript. The cloned gene was used to construct a null mutant to determine the role of ROX1 in regulating the expression of several heme-regulated genes. Constitutive expression of ANB1 (a heme-repressed gene) was observed in the null strain, indicating that ROX1 codes for a repressor or a facilitator of repression. Enhancement of expression of CYC7 in the null strain indicated that the ROX1 factor is required for repression of CYC7 to its normal low level of expression, consistent with evidence that CYC7 has a hybrid heme-induced, heme-repressed regulatory mechanism. The null mutation had only a slight negative effect on expression of the heme-induced genes CYC1 and tr-1 (a heme-induced homolog of ANB1), suggesting that the ROX1 factor is not directly involved in their regulation despite the existence of an unusual rox1 mutation (rox1-a1) causing constitutive expression of this group. The respiratory competence of the null mutant indicates that ROX1 is not a respiratory factor. ROX1 expression was found to be induced by heme, indicating that the heme repression of ANB1 and its family is the result of a cascade in which heme induces a repression factor which keeps the family of heme-repressed genes inactive during aerobic growth. The rox1-a1 allele had earlier been shown to cause constitutive expression of the family of heme-induced respiratory genes. This allele was found to cause constitutive expression of the ROX1 transcript itself, indicating that ROX1 is in the major heme-induced regulon.

Alleles↗

PubMed1988

Oxygen-dependent upstream activation sites of Saccharomyces cerevisiae cytochrome c genes are related forms of the same sequence.

In Saccharomyces cerevisiae, the two genes, CYC1 and CYC7, that encode the isoforms of cytochrome c are expressed at different levels. Oxygen regulation is mediated by the expression of the CYP1 gene, and the CYP1 protein interacts with both CYC1 upstream activation sequence 1 (UAS1) and CYC7 UASo. In this study, the homology between the CYP1-binding sites of both genes was investigated. The most noticeable difference between the CYC1 and CYC7 UASs is the presence of GC base pairs at the same positions in a repeated sequence in CYC7 compared with CG base pairs in CYC1. Directed mutagenesis changing these GC residues to CG residues in CYC7 led to CYC1-like expression of CYC7 both in a CYP1 wild-type strain and in a strain carrying the semidominant mutation CYP1-16 which reverses the oxygen-dependent expression of the two genes. Our results strongly support the hypothesis that the CYP1-binding sites in CYC1 and CYC7 are related forms of the same sequence and that the CYP1-16 protein has altered specificity for the variant forms of the consensus sequences in both genes.

Base Sequence↗

PubMed1987

Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes.

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

Base Sequence↗

PubMed1987

Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene.

The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein. Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300. In this study, the nature of the positive site and its relationship to the negative site has been investigated. Expression of the CYC7 gene is weakly inducible by oxygen. This effect was greatly enhanced by the semidominant CYP1-16 mutation in the trans-acting gene CYP1. The weak oxygen regulation in wild-type cells and the enhanced induction in CYP1-16 mutants were found to be mediated through the positive site. A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site. The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site. The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene. Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression. A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.

Cytochrome c Group↗

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

Oxygen regulation of anaerobic and aerobic genes mediated by a common factor in yeast.

The expression of a number of yeast genes is regulated by oxygen levels. While many of these are known to be induced in the presence of oxygen, we have described a gene, ANB1, that responds in the opposite fashion, being expressed only under anaerobic conditions. To identify genes involved in regulation of ANB1 and other oxygen-regulated genes, we selected mutations causing constitutive expression of ANB1, using a fusion of the ANB1 modulator segment to the CYC1 gene. A number of trans-acting mutations affecting a gene designated ROX1 caused constitutive expression of both the fused and wild-type genes, indicating that the ROX1 gene product operates through the ANB1 modulator sequence at the level of transcription. The mutant alleles of ROX1 fall into two phenotypic classes. The rox1-a class is semi-dominant, and the rox1-b class is recessive. One mutant, rox1-a1, is pleiotropic and causes constitutive expression of three oxygen-induced genes--CYC1, SOD (superoxide dismutase), and tr-1 (an oxygen-induced gene with homology to ANB1)--as well as constitutive expression of the oxygen-repressed ANB1 gene. Alleles of the rox1-b class cause constitutive expression of ANB1 but do not affect expression of the oxygen-induced genes tested. The pleiotropy of the rox1-a1 mutant indicates that the ROX1 gene product is involved in coordinate expression of both oxygen-induced and oxygen-repressed genes.

Aerobiosis↗

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↗

PubMed1984

Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.

Base Sequence↗

PubMed1983

The expression in yeast of the Escherichia coli galK gene on CYC1::galK fusion plasmids.

A set of gene fusions have been constructed between the transcriptional and translational initiation signals of the yeast CYC1 gene, encoding iso-1-cytochrome c, and the coding sequence of the Escherichia coli galK gene encoding galactokinase. These fusions are contained on plasmids which have both yeast and E. coli replication origins and selectable markers and, therefore, can be used to transform either yeast or E. coli cells. When galactokinase-deficient (gall-) yeasts were transformed with these plasmids the resulting Gal+ transformants were heterogeneous with respect to their galactokinase levels. The galactokinase levels in all were subject to glucose repression, characteristic of the transcriptional regulation of the CYC1 gene. The fusion points for representative plasmids were determined by DNA sequence analysis, and from these data, the differential expression of the galK gene could be explained. One fusion plasmid, YRpR1, which gave the highest level of galK expression, was characterized further. As an additional demonstration that galactokinase expression from the fusion was under CYC1 transcriptional control, a cis-dominant, CYC1-linked mutation known to drastically reduce CYC1 gene transcription was introduced into YRpR1 and shown to similarly effect galK expression. The galK mRNA produced from the fused gene of YCpR1, a centromere-containing derivative of YRpR1, consisted of the mRNA leader sequence plus the first four codons of the CYC1 gene, the galK coding sequence, then the remainder of the CYC1 coding sequence and the 175 nucleotide non-translated 3' sequence. As a demonstration of the usefulness of these plasmids for the selection of regulatory mutants, two mutants capable of greatly enhanced levels of galactokinase expression were isolated. Preliminary characterization of these mutations indicates that they likewise affect the expression of the chromosomal CYC1 gene.

Anaerobiosis↗

PubMed1983

Modulator sequences mediate oxygen regulation of CYC1 and a neighboring gene in yeast.

Three transcripts from Saccharomyces cerevisiae--CYC1 mRNA (transcribed from the iso-I cytochrome c gene) and two RNAs of unknown function, designated tr-1 and tr-2-were identified by reverse Southern blot analysis and found to be regulated in response to oxygen. CYC1 mRNA and tr-1 accumulation occurred only in the presence of oxygen while tr-2 appeared only under anaerobic conditions. tr-2 was transcribed from a region approximately 1 kilobase 5' from the CYC1 coding sequence and in the opposite direction. tr-1 showed homology to the same region as tr-2 but was transcribed from elsewhere in the genome. Expression of tr-2 and CYC1 was observed to be normal in cells transformed with centromeric plasmids carrying the two genes. Mutant transforming plasmids were constructed in which a 400-base-pair region between tr-2 and CYC1 was either deleted or inverted. The deletion led to low-level nearly unregulated expression of both the CYC1 and tr-2 genes, suggesting that sequences upstream from both genes are important for their expression and regulation. The inversion mutation produced a reversed pattern of CYC1 regulation in which the mRNA was present in anaerobically grown cells but absent in the presence of oxygen, mimicking wild-type tr-2 regulation and suggesting that the CYC1 transcription unit is under the control of the translocated tr-2 modulator sequences. Models for the function of these modulators are discussed.

Cytochrome c Group↗

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↗

PubMed1982

The molecular characterization of three transcriptional mutations in the yeast iso-2-cytochrome c gene.

Three mutations, each of which causes overproduction of iso-2-cytochrome c, were characterized biochemically. Two, CYP3-4 and CYP3-15, were previously shown to be cis-dominant and map to the CYC7 locus which encodes the iso-2 protein, while the third, cyp1-16, maps to an unliked locus. All three mutations caused dramatically increased levels of transcription of the CYC7 gene, and the CYC7 mRNA in mutant cells was found to be the same size as that in wild type cells. The CYP3-4 mutation was found to be caused by the integration of a transposable element, Tyl, 269 base pairs 5' to the coding sequences. The CYP3-15 mutation was also found to alter the DNA, probably through a deletion or inversion with one endpoint 285 base pairs upstream from the coding sequence. The CYC7 gene in both wild type and mutant cells was not subject to catabolite repression.

Base Sequence↗

PubMed1981

Sequence of the yeast iso-1-cytochrome c mRNA.

The nucleotide sequence of the yeast iso-1-cytochrome c (CYC1) mRNA is presented. The mRNA was enriched by hybridization to cloned CYC1 DNA attached to a solid matrix: either nitrocellulose filters or diazobenzyloxymethyl cellulose powder. The sequence of the 5'-end of the mRNA was determined by the extension of a CYC1-specific dodecanucleotide primer; the sequence of the 3'-end was determined using a decanucleotide d(pT8-G-A) primer. The CYC1 mRNA begins 61 nucleotides 5' to the AUG initiation codon, extends through the coding sequence to 172 to 175 nucleotides 3' to the UAA termination codon, followed by the poly(A) tail. There are no intervening sequences. Some of the sequences that the CYC1 mRNA shares in common with other eukaryotic mRNAs are discussed.

Base Sequence↗