Biomedical subjects

C V Lowry

Publications and source records attributed to C V Lowry.

29 records · Page 2Linked to original sources

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↗

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

Metabolite changes in individual rat muscle fibers during stimulation.

Rat plantaris and soleus muscles were stimulated intensely in vivo for 1 and 15 min, freeze-clamped, and freeze-dried, and individual fibers were dissected free. Fibers, assigned to four groups on the basis of lactate dehydrogenase and malate dehydrogenase, were each separately analyzed for ATP, P-creatine, glycogen, glucose, glucose-6-phosphate (glucose-6-P), lactate, citrate, and malate. Some fibers were also analyzed for fructose 1,6-phosphate, total adenylate and total creatine. Although each group as a whole showed significant and often large differences in control composition and response to stimulation, individual fibers varied enough to create an almost continuous spectrum of metabolite levels from one extreme to the other. The data suggest that the slowest twitch fibers were the most active in the control state. Stimulation for 1 min caused a small increase in ATP in all groups with a large decrease in P-creatine in "fast white" fibers and a modest decrease in the rest. After 15-min stimulation, fast white fibers had lost 60% of initial ATP and 97% of initial P-creatine, whereas in other fiber types these compounds underwent little further change. Metabolite changes with stimulation were also greatest in fast white fibers. Glucose-6-P rose 15-fold in 1 min, then fell to below control by 15 min when glycogen had been exhausted; lactate rose two to six times more than in other types. Glucose rose in all groups to levels at 15 min, compatible with equilibrium with blood plasma.

Adenosine Triphosphate↗

PubMed1980

Enzyme levels in individual rat muscle fibers.

Individual muscle fibers from the rat anterior tibialis and soleus muscles were each analyzed in duplicate for lactate dehydrogenase (LDH, EC 1.1.1.27), malate dehydrogenase (MDH, EC 1.1.1.37), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), fumarate hydrotase (EC 4.2.1.2), glycogen phosphorylase (EC 2.4.1.1), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), fructose-bisphosphatase (EC 3.1.3.11), and creatine kinase (EC 2.7.3.2). A few fibers were also analyzed for adenylate kinase (EC 2.7.4.3). In general, there was a wide and almost continuous spectrum of coordinated enzyme activities. In the tibialis muscle, two fiber groups could be clearly distinguished on the basis of MDH activity. The high MDH group had on the average lower LDH activity, but there was a great deal of overlap in LDH between the two groups. Less overlap was observed for phosphorylase and fructose-bisphosphatase, both inversely related to MDH. Only one main group of fibers (presumably slow twitch) was found in the soleus muscle, although enzyme activities also covered a wide range. These soleus fibers were clearly distinguished from the high MDH tibialis group by much lower activities of LDH, pyruvate kinase, and fructose-bisphosphatase.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1978