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R C Dickson

Publications and source records attributed to R C Dickson.

At least 19 recordsLinked to original sources
PubMed1992

Glycine cytoprotection during lethal hepatocellular injury from adenosine triphosphate depletion.

Glycine protects renal tubule cells from cell death during adenosine triphosphate (ATP) depletion. Although the liver plays a key role in glycine metabolism, information is lacking regarding the effects of glycine on lethal hepatocellular injury. Thus, the aim of this study was to determine the potential cytoprotective role of glycine during ATP depletion of rat hepatocytes. Metabolic inhibition with 2.5 mmol/L potassium cyanide (KCN) was used to produce ATP depletion. Hepatocyte suspensions treated with KCN had a 2-hour viability of 5.9% +/- 2.0%, whereas cells treated with KCN in the presence of 2.0 mmol/L glycine had a viability of 80.2% +/- 1.5%, which was virtually identical to controls (81.5% +/- 1.9%). Glycine cytoprotection was dose dependent and amino acid specific. The cytoprotective effect of glycine was not mediated by protein synthesis, glycine mitochondrial metabolism, cytosolic acidosis, or preservation of either intracellular cellular glutathione or ATP. However, glycine did decrease total cellular proteolysis by 18% +/- 2%, 25% +/- 3%, and 33% +/- 1% after 1, 2, and 3 hours of KCN treatment, respectively (P less than 0.01). Inhibition of proteolysis by glycine was dose dependent over the same range as its cytoprotection. The results suggest that glycine protects against hepatocellular injury by inhibiting degradative proteolytic activity. It was concluded that proteolysis may be an important mechanism contributing to lethal injury of hepatocytes during ATP depletion.

Adenosine Triphosphate↗
PubMed1992

Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae.

To study sphingolipid function(s) in Saccharomyces cerevisiae, we have investigated the effects of environmental stress on mutant (SLC) strains (R. C. Dickson, G. B. Wells, A. Schmidt, and R. L. Lester, Mol. Cell. Biol. 10:2176-2181, 1990) that either contain or lack sphingolipids, depending on whether they are cultured with a sphingolipid long-chain base. Strains lacking sphingolipid were unable to grow at low pH, at 37 degrees C, or with high salt concentrations in the medium; these environmental stresses are known to inhibit the growth of some S. cerevisiae strains with a defective plasma membrane H(+)-ATPase. We found that sphingolipids were essential for proton extrusion at low pH and furthermore found that cells lacking sphingolipid no longer exhibited net proton extrusion at normal pH after a 1-min exposure to pH 3. Cells lacking sphingolipid appeared to rapidly become almost completely permeable to protons at low pH. The deleterious effects of low pH could be partially prevented by 1 M sorbitol in the suspension of cells lacking sphingolipid. Proton extrusion at normal pH (pH 6) was significantly inhibited at 39 degrees C only in cells lacking sphingolipid. Thus, the product of an SLC suppressor gene permits life without sphingolipids only in a limited range of environments. Outside this range, sphingolipids appear to be essential for maintaining proton permeability barriers and/or for proton extrusion.

Fungal Proteins↗
PubMed1992

Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection.

A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, lcb1 and lcb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCB1 and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb- strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of alpha-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chain-base starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb- S. cerevisiae and inositol starvation of Inos- S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis.

Cell Division↗
PubMed1992

The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

Alleles↗
PubMed1991

Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains.

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.

Amino Acid Sequence↗
PubMed1991

Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids.

The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.

Acyltransferases↗
PubMed1991

Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

Base Sequence↗
PubMed1990

The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster.

The DNA binding domain of the transcription factor LAC9 contains 6 cysteine residues with spacing in the primary peptide sequence identical to that found in the DNA binding domain of the GAL4 transcription factor. In GAL4, the CysX2CysX6CysX6CysX2CysX6Cys motif has been shown to form a Zn(II)2Cys6 binuclear cluster (Pan, T. and Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2077-2081), representing a new structure for a Zn(II)-containing transcription factor which differs from the "zinc finger" motif first described for TFIIIA. LAC9 has been shown to bind two Zn(II) ions (Halvorsen, Y. C., Nandabalan, K., and Dickson, R. D. (1990) J. Biol. Chem. 265, 13283-13289). The similarity of the amino acid sequence and the Cys spacing within the DNA binding domain suggest that LAC9 should also be capable of forming the Zn(II)2Cys6 cluster found in GAL4. A fragment of LAC9 consisting of 144 amino acid residues spanning the DNA binding domain has been prepared with 113Cd(II) substituted for the two native Zn(II) ions. 113Cd NMR of this fragment (denoted LAC9(85-228*] has been carried out in an attempt to test the hypothesis that LAC9, like GAL4, forms a binuclear cluster. The chemical shifts of the two bound 113Cd(II) ions, 705 and 692 ppm respectively, are consistent with ligation of each 113Cd(II) ion to 4 sulfur atoms. The best model for such ligation is that two of the cysteine S- form bridges between the two Cd(II) ions. Formation of a Zn(II)-Cd(II) hybrid form of LAC9(85-228*) has also been observed. We conclude that LAC9 contains a Zn(II)2Cys6 binuclear cluster as previously reported for GAL4.

Cations, Divalent↗
PubMed1990

Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9.

The GAL4 protein of Saccharomyces cerevisiae and the LAC9 protein of Kluyveromyces lactis are transcription activator proteins with similar structure and function. Greatest similarity occurs in the C region near the carboxy terminus, where 16 of 18 amino acids are identical. The function of the C region is unclear. Here we show that the structural similarity is reflected in functional similarity. Single amino acid changes in the C region of GAL4 and LAC9 create a similar phenotype: constitutive gene expression. In S. cerevisiae the constitutive phenotype caused by GAL4 mutants can be abolished by overproduction of GAL80. These results support a model in which the C region of GAL4 and LAC9 constitute similar negative regulatory domains that interact with GAL80 in S. cerevisiae and an unidentified GAL80 homolog in K. lactis. This protein-protein interaction prevents expression of the galactose operon in the uninduced state.

Amino Acid Sequence↗
PubMed1990

LAC9 DNA-binding domain coordinates two zinc atoms per monomer and contacts DNA as a dimer.

The LAC9 protein of Kluyveromyces lactis activates transcription by binding to upstream activating sequences lying in front of genes of the lactose-galactose regulon. LAC9 belongs to a family of fungal proteins having a conserved domain containing 6 cysteines. This domain, termed a C6 zinc finger, is thought to bind one zinc atom and to play a vital role in DNA binding. To further characterize the DNA-binding domain of LAC9, we have developed a procedure to produce and to purify milligram amounts of LAC9 peptides. The two larger peptides, one containing amino acids 1-228 and the other containing amino acids 85-228, formed dimers in solution and bound DNA specifically as a dimer. The smallest LAC9 peptide, amino acids 85-160, failed to dimerize and did not bind DNA. Atomic absorption spectroscopy revealed that each LAC9 monomer coordinated two zinc atoms, not one, as had been predicted. This result suggests, as does previously published data, that the C6 zinc finger domain has a unique conformation that may represent a new type of DNA-binding motif.

Cloning, Molecular↗
PubMed1990

The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Amino Acid Sequence↗
PubMed1990

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

Blotting, Southern↗
PubMed1988

Primary structure of the lactose permease gene from the yeast Kluyveromyces lactis. Presence of an unusual transcript structure.

The LAC12 gene of Kluyveromyces lactis codes for an inducible lactose permease. We have determined the nucleotide sequence of a DNA fragment which includes the complete LAC12 gene. The 4.7-kilobase (kb) mRNA carrying LAC12 contained two open reading frames, ORFI (1761 bases) and ORFII (1266 bases), separated by a 573-base pair noncoding region. Mung bean and exonuclease VII mapping showed that there was no splicing of the 4.7-kb transcript and thus no intron between the two open reading frames. Chromosomal disruption of ORFI with the URA3 gene destroyed lactose transport activity, suggesting that ORFI codes for a component of the permease. Disruption of ORFII and the noncoding region between the two open reading frames did not affect the lactose permease function, indicating that they do not comprise a part of the permease. We do not know if ORFII is translated, but in either case, the structure of the 4.7-kb mRNA is unusual. We discuss possible origins for it. The peptide predicted from ORFI is hydrophobic as would be expected for a membrane-bound protein. Compared with other membrane proteins, LAC12 (ORFI) protein showed sequence similarity to the human glucose and the Escherichia coli xylose-H+ and arabinose-H+ transporters. No obvious amino acid sequence similarity was found with the lactose permease of E. coli.

Amino Acid Sequence↗
PubMed1988

The organization and transcription of the galactose gene cluster of Kluyveromyces lactis.

The yeast Kluyveromyces lactis grows on galactose by inducing the Leloir pathway enzymes-kinase, epimerase, and transferase. To investigate the molecular mechanism for regulating expression of this metabolic pathway we isolated GAL1, GAL7, GAL10, which code for kinase, transferase, and epimerase, respectively, and characterized their size, organization, and transcriptional regulation. Our results indicate that induction of the Leloir pathway in K. lactis occurs at the level of transcription and that the organization and regulation of the GAL gene cluster in K. lactis is closely related to the homologous gene cluster in Saccharomyces cerevisiae. Likewise, the Upstream Activator Sequences that regulate induction of the GAL genes are similar in base sequence, number and relative location in the two yeasts.

Cloning, Molecular↗
PubMed1988

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of mutants of Kluyveromyces lactis defective in lactose transport.

Mutants of Kluyveromyces lactis defective in lactose transport were identified among lactose-resistant revertants of lactose-sensitive strains. The mutations are closely linked to the beta-galactosidase gene, LAC4, and they are located in a previously identified gene, LAC12, which has been shown to code for a lactose permease. Our data establish that LAC12 is the only lactose permease gene in K. lactis. The lactose permease also transports galactose. LAC12 is transcribed in a direction opposite to that of LAC4, there being about 2.5 kb between their transcription start sites. Transcription of LAC12 is inducible as is that of all other structural genes in the lactose-galactose regulon of K. lactis.

Biological Transport↗