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Biomedical subjects

A P James

Publications and source records attributed to A P James.

At least 19 recordsLinked to original sources

Re-feeding after starvation involves a temporal shift in the control site of glycogen synthesis in rat muscle.

The starved-to-fed transition is accompanied by rapid glycogen deposition in skeletal muscles. On the basis of recent findings [Bräu, Ferreira, Nikolovski, Raja, Palmer and Fournier (1997) Biochem. J. 322, 303-308] that during recovery from exercise there is a shift from a glucose 6-phosphate/phosphorylation-based control of glycogen synthesis to a phosphorylation-based control alone, this paper seeks to establish whether a similar shift occurs in muscle during re-feeding after starvation in the rat. Chow re-feeding after 48 h of starvation resulted in glycogen deposition in all muscles examined (white, red and mixed quadriceps, soleus and diaphragm) to levels higher than those in the fed state. Although the early phase of re-feeding was associated with increases in glucose 6-phosphate levels in all muscles, there was no accompanying increase in the fractional velocity of glycogen synthase except in the white quadriceps muscle. This finding, together with the observation that the fractional velocity of glycogen synthase in most muscles was already high in the starved state, suggests that in the initial phase of glycogen deposition the phosphorylation state of the enzyme may be adequate to support net glycogen synthesis. In the later phase of re-feeding, the progressive decrease in the fractional velocity of glycogen synthase in association with a decrease in the rate of glycogen deposition suggests that glycogen synthesis is controlled primarily by changes in the phosphorylation state of glycogen synthase. In conclusion, this study suggests that there is a temporal shift in the site of control of glycogen synthesis as glycogen deposition progresses during re-feeding after starvation.

Animals

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants.

Plasmid YEp ( ADE1 )1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI -1 (Morris et al., 1981), results in high frequency, unstable transformation of ade 1 yeast strains. A second plasmid, YRp ( ADE1 )2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade 1 strains by hybridization analysis, and (3) a transformant carrying a multimeric form of YRp ( ADE1 )2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade 1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.

Cloning, Molecular

Genetic system of Schwanniomyces alluvius determined by diad analysis of fusion products.

The genetics of Schwanniomyces alluvius, a yeast that secretes alpha-amylase, were investigated. No mating types have been detected in this haploid organism. Hybrids were produced by protoplast fusion, and these were subjected to diad analysis by using two-spored asci. Results showed that the diploidy introduced by cell fusion persists through successive spore generations. It was concluded that in this organism, sporulation is preceded by the fusion of mitotic products, regardless of the ploidy of the latter. Routine procedures for constructing novel strains would, no doubt, be hampered by this failure of the sporulation process to restore haploidy. Nevertheless, chromosomal instability of hybrids, as indicated both by heterogeneity of fusion products and by a high frequency of morbidity among their segregants, may permit the use of classical genetic techniques for strain construction.

Ascomycota

Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.

A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.

Agglutination

Nuclear mutations in Saccharomyces cerevisiae conferring multiple sensitivity to petite-inducing treatments.

Eighteen EMS-induced mutant strains of S. cerevisiae with increased sensitivity to petite induction by sulfanilamide-aminopterin treatment have been isolated. Four of these strains demonstrated a concomitant increase in sensitivity to the petite-inducing effects of u.v. irradiation and of growth at an elevated temperature. Of these, two were shown to be a consequence of recessive nuclear mutations at one gene (sas1-1 and sas1-2). Expression of the two remaining mutations was too low to permit genetic analysis. All three petite-inducing treatments used in this study are known to reduce the hydrogen bond strength between the two strands of double stranded DNA, and the existence of multiple sensitivity is discussed in terms of an alteration of a mitochondrial endonuclease which acts preferentially at sites of reduced attraction.

Aminopterin

Genetic analysis of Saccharomyces cerevisiae transformed by plasmid containing a supressor transfer ribonucleic acid gene.

The behavior in Saccharomyces cerevisiae of plasmid pYTE1, which contains yeast tyrosine-inserting ochre suppressor SUP4.o, a 4-kilobase EcoRI fragment of yeast 2muDNA, and the bacterial plasmid pBR322, has been studied. Selection of yeast transformants was by suppression of multiple ochre mutations. About 10(3) to 10(4) transformants per microgram of pYTE1 dfeoxyribonucleic acid were obtained. The majority of transformants contained both an integrated copy of the SUP4.o gene plus pBR322 deoxyribonucleic acid sequences and autonomously replicating forms of the plasmid. The integrated copy was extremely stable mitotically and meiotically, but the associated nonintegrated copies were lost at meiosis. The chromosomally integrated pBR322 sequences were linked to the SUP4.o gene. The integration site was at the SUP4+ locus. In transformants with only nonintegrated copies of pYTE1, the expression of suppression was reduced, and the plasmid was unstable in mitosis. Plasmid deoxyribonucleic acid preparations from both types of transformant could be used to retransform yeast cells. Plasmid pYTE1 has restriction enzyme sites useful for the high frequency and stable transformation of other genes into yeasts. The potential uses of this plasmid for transformation of other organisms is discussed.

Genetic Linkage

The mutagenic potential of unexcised pyrimidine dimers in Saccharomyces cerevisiae, rad1-1: evidence from photoreactivation and pedigree analysis.

Photoreactivation and pedigree analysis have been combined to show that unexcised pyrimidine dimers in the DNA of rad1-1 yeast can initiate mutagenesis after passing through several DNA replications. Monomerisation of dimers immediately before the second replication to follow UV has no effect on mutants appearing after the first post-UV cell division but reduces second-generation mutants to one third of their frequency in the dark and has a similar through slightly less marked effect on mutants appearing in the third or subsequent generations. The bearing of these findings on the mechanism of UV mutagenesis is dicussed.

DNA

The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation.

The mechanism of UV-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61 percent at survival levels of 90 and 77 percent, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective.

Cell Division

Alterations in mitochondrial DNA of yeast which accompany genetically and environmentally controlled changes in rho- mutability.

Alterations in the physical characteristics of mitochondrial DNA accompanied increased spontaneous mutability to cytoplasmic respiratory-deficiency in yeast. Two systems were used to modify mutation rates, one physiological, the other genetic. Cells in log phase were shown to be more mutable than cells in stationary phase, and glucose-repressed cells were shown to be more mutable than unrepressed cells. A nuclear gene which acts as a mitochondrial mutator was found to increase spontaneous mutation rate by a factor of ten. An increase in endogenous formation of G+G-rich fragments of mt-DNA accompanied a physiological state conducive to higher mutability, and it is proposed that increased in vivo digestion of A+T-rich regions is involved in these alterations. Greater nuclease(s) activity accompanied the presence of the mutator gene, and it is proposed that this gene is concerned with the regulation of nuclease activity or with repair mechanisms.

Centrifugation, Isopycnic

Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity.

A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.

Ascomycota

Evidence of preferential pairing of chromosomes at meiosis in aneuploid yeast.

Meiotic pairing in homothallic S. cerevisiae was studied by tetrad analysis, using strains that were trisomic or tetrasomic for chromosomes. I. The disomic segregants of these strains produce tetrasomic spore colonies that can be distinguished by their phenotype. Results indicated the existence of preferential pairing and nonrandom assortment of chromosomes at meisosis I. The frequency of crossing over is apparently normal in at least some regions when nonpreferred pairing occurs.

Adenine

A kinetic analysis of spontaneous rho- mutations in yeast.

Spontaneous mutation to the petite state at the level of the individual cell was studied in a haploid strain of yeast by the technique of pedigree analysis. Results indicated that (1) the mutability of rho+ cells within a population in log phase is variable; (2) rho+ mitotic buds are, on the average, about 50% more mutable than the rho+ cells from which they arose; (3) the mutability of a rho+ cell tends to decrease as it produces consecutive buds: (4) the probability that a mother cell will become rho- at or immediately subsequent to cell division is, on the average, one third the probability that its bud will be rho-; (5) most, if not all spontaneous rho- mutant cells contain mitochondrial DNA as judged from suppressiveness measurements. The data indicate that the spontaneous production of a mutant cell is a multi-step process. Neither a replicative advantage of defective mitochondrial DNA nor the existence of a "master" mitochondrial genome provides a satisfactory explanation of the process. Either selective dispensation of defective mitochondria to the bud at cytokinesis or normal retention by the mother cell of factors influencing the amplification or rate of induction of defective mitochondrial DNA could be involved.

Extrachromosomal Inheritance

Lethal sectoring and the delayed induction of aneuploidy in yeast.

Persistent lethal sectoring in a homothallic strain of yeast has been ascribed to tetrasomy for chromosome I. Such aneuploids can appear many generations after irradiation. The data thus indicate that an induced predisposition towards aneuploidy can be prolonged through successive post-irradiation cell divisions. Sporadic cell death in tetrasomics for chromosome I was found to result from a metabolic imbalance and not from a genetic instability conseqent to aneuploidy. This imbalance may be due to a dosage effect involving cistrons for ribosomal RNA since many of these are known to be located on chromosome I. Tetrasomy is not the only cause of persistent lethal sectoring; the phenomenon has been initiated through genetic recombination involving normal diploids. It has also been concluded that, in trisomics, equational division of the supernumerary chromosome sometimes occurs at the first meiotic division.

Aneuploidy