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T G Cooper

Publications and source records attributed to T G Cooper.

At least 235 records · Page 13Linked to original sources
PubMed1984

Nucleotide sequence of the Saccharomyces cerevisiae arginase gene (CAR1) and its transcription under various physiological conditions.

We have determined the nucleotide sequence of the yeast CAR1 gene along with its 5' and 3' flanking sequences. The structural portion of this gene encodes a protein of 333 amino acid residues with a calculated Mr value of 35,616. The transcripts produced from this gene are modestly heterogeneous at their 5' and 3' termini. Most 5' termini map to a position 42 to 49 base pairs upstream from the ATG at the beginning of the open reading frame. The 3' termini map to a position 108 to 127 base pairs downstream of the amber codon which terminates the open reading frame. A variety of potentially significant sequences were identified in the regions flanking the structural portion of the gene.

Amino Acid Sequence↗
PubMed1983

What is the function of nitrogen catabolite repression in Saccharomyces cerevisiae?

In contrast to the previously held notion that nitrogen catabolite repression is primarily responsible for the ability of yeast cells to use good nitrogen sources in preference to poor ones, we demonstrate that this ability is probably the result of other control mechanisms, such as metabolite compartmentation. We suggest that nitrogen repression is functionally a long-term adaptation to changes in the nutritional environment of yeast cells.

Allantoin↗
PubMed1983

Oxalurate induction of multiple URA3 transcripts in Saccharomyces cerevisiae.

The URA3 gene from Saccharomyces cerevisiae is localized on a 1.1-kilobase (kb) DNA fragment. By using this fragment as a hybridization probe, we found that oxalurate, a gratuitous inducer of the allantoin degradative system, also serves to induce URA3 specific RNA. This response is restricted to oxalurate; other conditions which bring about high-level synthesis of the allantoin degradative enzymes did not produce the effect. Two classes of RNA (1.0 and 1.5 kb) were found to be oxalurate induced. Both classes are encoded by the URA3 gene, overlap, and probably do not significantly differ at their 5' termini. Northern blot mapping of the transcripts indicated that the 1.5-kb transcript was likely encoded by sequences extending up to 0.5 kb downstream from the 3' terminus of the 1.0-kb transcript. Analysis of the endpoints of the major 1.0-kb URA3 transcript by S1 nuclease mapping revealed the existence of two 5' termini, separated by 5 to 10 nucleotides, and seven 3' termini, separated by 5 to 20 nucleotides each, over a range of about 70 bases.

Amino Acids↗
PubMed1982

Stroboscopic illumination for the assessment of hyperactivated motility of mouse spermatozoa.

Spermatozoa showing hyperactivated motility can be recognized in multiple-exposure micrographs. The cells were confined in 10-micrometer-deep chambers, illuminated in darkfield conditions, and photographed using a stroboscope (10 Hz). Hyperactivation, as judged from the pattern of ten superimposed images, could be promoted by raising the ionic strength of the medium but did not occur in the absence of calcium.

Animals↗
PubMed1982

Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast.

Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits.

Allophanate Hydrolase↗
PubMed1982

Ultrastructure of the perfused rat epididymis: effect of luminal sodium ion concentration.

The appearance of the rat epididymal epithelium changed when it was perfused in vivo through the lumen with unphysiologically high sodium ion concentrations; dilatation of intercellular spaces (ICS) at threshold concentrations of 30 mM-Na+ in the cauda and about 55 mM-Na+ in the corpus was associated with absorption of water from the lumen. Despite the distended ICS, junctional complexes appeared intact, and their integrity was confirmed by the exclusion of luminal horseradish peroxidase (HRP) from the ICS, and by demonstrating that circulating [3H]inulin did not enter the lumen. Smooth ER and lipid droplets in the principal cells of the corpus epididymidis were well maintained, and the preservation of granular ER in principal cells of the cauda epididymidis lent morphological support to the continued secretion of protein in this segment. However, occasional distension or involution of inner Golgi cisternae was evident in principal cells after 3-6 h perfusion. In contrast to multivesicular bodies of principal cells, the apical and basal vacuoles characteristic of clear cells changed in size with different perfusion. In contrast to multivesicular bodies of principal cells, the apical and basal vacuoles characteristic of clear cells changed in size with different perfusing solutions. When low Na+ concentrations were perfused large translucent vacuoles were frequently found in the apical cytoplasm of clear cells in the corpus and cauda epididymidis, and filled vacuoles became larger and showed a decrease in content density in the cauda epididymidis. These large vacuoles were absent from tissue perfused with high Na+ concentrations. Normal pinocytotic activity of both cell types was demonstrated by perfusing HRP which was taken up by the normal route in principal cells, with some transfer to the Golgi cisternae. By far the most HRP was accumulated in clear cell vacuoles irrespective of the composition of the perfusing solution.

Absorption↗
PubMed1982

Pleiotropic control of five eucaryotic genes by multiple regulatory elements.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.

Allantoin↗
PubMed1982

Isolation and subcloning analysis of functional centromere DNA (CEN11) from Saccharomyces cerevisiae chromosome XI.

We have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Genetic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also contained on the segment of S. cerevisiae DNA cloned in pYe(MET14)2. This plasmid is maintained stably in budding S. cerevisiae cultures and segregates predominantly 2+:20- through meiosis. The CEN11 element has been subcloned in vector YRp7' on an S. cerevisiae DNA fragment 900 base pairs in length [pYe(CEN11)10]. The mitotic and meiotic behavior of plasmids containing CEN11 plus a DNA replicator (ars) indicates that the centromere DNA sequences enable these plasmids to function as true minichromosomes in S. cerevisiae.

Base Sequence↗
PubMed1982

Isolation of the CAR1 gene from Saccharomyces cerevisiae and analysis of its expression.

We isolated the CAR1 gene from Saccharomyces cerevisiae on a recombinant plasmid and localized it to a 1.58-kilobase DNA fragment. The cloned gene was used as a probe to analyze polyadenylated RNA derived from wild-type and mutant cells grown in the presence and absence of an inducer. Wild-type cells grown without the inducer contained very little polyadenylated RNA capable of hybridizing to the isolated CAR1 gene. A 1.25-kilobase CAR1-specific RNA species was markedly increased, however, in wild-type cells grown in the presence of inducer and in constitutive, regulatory mutants grown without it. No CAR1-specific RNA was observed when one class of constitutive mutant was grown in medium containing a good nitrogen source, such as asparagine. Two other mutants previously shown to be resistant to nitrogen repression contained large quantities of CAR1 RNA regardless of the nitrogen source in the medium. These data point to a qualitative correlation between the steady-state levels of CAR1-specific, polyadenylated RNA and the degree of arginase induction and repression observed in the wild type and in strains believed to carry regulatory mutations. Therefore, they remain consistent with our earlier suggestion that arginase production is probably controlled at the level of gene expression.

Arginase↗
PubMed1982

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.

Allophanate Hydrolase↗
PubMed1982

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.

Allantoin↗
PubMed1982

Secretion of inositol and glucose by the perfused rat cauda epididymidis.

Inositol was detected by gas-liquid chromatography in fluid perfused at 1.3 microliter/min through the lumen of the distal cauda epididymidis of anaesthetized rats. The concentration (247 microM) exceeded that in blood (48 microM) and the secretion rate was constant for 5 h. D-[3H]Myo-inositol infused for 3 h into the general circulation of rats (1 muCi/min) also appeared in fluid perfusing the lumen, whether or not 50 mM-inositol was present in the perfusing solution. No plateau of radioactivity was reached during infusion, and by the end of 3 h perfusate activity was 26% of that in blood. Calculation of the specific activity of inositol in perfusates and blood plasma suggested that blood was not the immediate source of luminal inositol, and that any endogenous pool of cyclitol has a turnover time of greater than 3 h. [3H]Myo-inositol perfused through the lumen was not absorbed by the tissue. These data suggest that the high concentration of inositol in epididymal fluid (49 mM) is derived in part by epididymal secretion from a pool that is only slowly replenished from blood, and maintained by the impermeability of the epithelium. Glucose also appeared in fluid perfused through the epididymal lumen, but its concentration (461 microM) was much less than in blood (8.5 mM), so this sugar may diffuse down a concentration gradient.

Animals↗
PubMed1981

Entry of glycerol into the rat epididymis and its utilization by epididymal spermatozoa.

The availability of glycerol in the epididymal lumen, and the extent of its utilization by spermatozoa, was studied by measurement of the substrate concentration in epididymal fluid and its oxidation in vitro. The source of luminal glycerol was sought by examining its penetration through the epididymal epithelium of a sperm-free tubule by the technique of luminal perfusion during intravenous infusion of glycerol. [3H]Glycerol was rapidly metabolized to a volatile and mobile molecule that quickly equilibrated between blood plasma and the epididymal lumen and so could not be used to monitor transfer. When blood levels of glycerol were raised by infusion of large amounts of the unlabelled compound, glycerol was detected in epididymal perfusates and a positive linear correlation existed between the rate of secretion into the epididymal lumen and the blood plasma concentration, suggesting that passive diffusion across the epithelium from blood could occur. In normal rats, however, the concentration of glycerol in sperm-free epididymal fluid (1.15 mM) exceeded that in blood plasma (0.35 mM). Luminal glycerol is therefore thought to arise from degradation of epididymal lipid.

Animals↗
PubMed1980

Structural analysis of the dur loci in S. cerevisiae: two domains of a single multifunctional gene.

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.

Allophanate Hydrolase↗
PubMed1980

The general importance of proteins and other factors in the transfer of steroids into the rat epididymis.

The entry of total radioactivity into the perfused tubule of the cauda epididymidis was measured during intravenous infusion of labelled steroids into the anaesthetised rat. The effect of altering the composition of the perfusate on this entry and the nature of radiometabolites was also studied. No radioactivity entered the epididymal lumen during infusions of cholesterol, and in the absence of luminal proteins the entry of isotope was generally low (< 14% plasma levels) for all steroids except dehydroepiandrosterone. C21/22-steroids exhibited a delay of about 30 min before a slow rise in luminal activity reached between 3% and 14% plasma levels. For C19-steroids a low plateau level of radioactivity was reached (at 6%-9% plasma levels) with dihydrotestosterone and androstenedione, whereas luminal activity continued to rise with respect to blood during infusions of dehydroepiandrosterone. When protein was added to the perfusing solution the delay in entry of radioactivity was decreased and plateau levels established for C21/22-steroids (at 14%-38% plasma levels). The extents of entry of dihydrotesterone and androstenedione were raised (34%-47%) and radioactivity continued to rise in the lumen relative to blood, but at a faster rate during infusions of dehydroepiandrosterone and perfusion with protein. Testicular fluid protein had little effect on the entry of activity for any steroid. There was no correlation between the extent of entry of tracer with binding of the steroids to plasma or perfusates, or partition into lipids. Tentative identification of metabolites indicated an entry of both dihydrotestosterone and dehydroepiandrosterone during their respective infusions. Androstenedione and androstanediols were excluded whereas 5 alpha-reduced metabolites appeared during of androstenedione and dehydroepiandrosterone.

Androgens↗