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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Entomologia experimentalis et applicata 60 (1991), S. 173-182 
    ISSN: 1570-7458
    Keywords: Genetics ; evolution ; host adaptation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When populations are exposed to different environments, evolutionary processes can lead either to genetically differentiated strains or to the appearance of increased generalism at the individual level. For evolution to occur, genetic variability in performance in different environments is required. Here, intraspecific genetic variation across environments was estimated in the flour beetle Tribolium castaneum (Herbst) by comparing the responses of two strains of T. castaneum to different flour types. Replicated groups from each strain were allowed to develop on either the standard whole wheat medium or on one of four novel flours (wheat, rice, corn and oat). In several of the novel flours, clear differences in mean development time or population size of one or both strains were seen relative to performance in the standard medium. Moreover, the strains differed significantly in their phenotypic responses to the flours. One strain did particularly poorly on oat flour. Reduced oviposition, reduced larval survivorship and increased larval cannibalism were examined as possible causes of the low productivity on oat flour. These three factors accounted for about 70% of the reduction in population size when this strain oviposited and developed in oat flour. The difference between these two outbred strains in response to these five flours suggests that genetic variation in resource use is present within T. castaneum and may also be present within strains and natural populations in grain storage facilities. Such variation would permit an evolutionary response to selection in multiple environments (flours). This process has agricultural implications when several types of grain are stored in a single location because it could eventually lead to the evolution of highly generalized populations of T. castaneum, an important pest of stored products.
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  • 2
    ISSN: 1572-8773
    Keywords: Iron transport ; Siderophores ; Pseudomonas putida ; Genetics ; Receptors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Root-colonizingPseudomonas putida WCS358 enhances growth of potato in part by producing under iron-limiting conditions a yellow-green, fluorescent siderophore designated pseudobactin 358. This siderophore efficiently complexes iron(III) in the rhizosphere, making it less available to certain endemic microorganisms, including phytopathogens, thus inhibiting their growth. At least 15 genes distributed over five gene clusters are required for the biosynthesis of pseudobactin 358. High-affinity iron(III) transport in strain WCS358 is initiated by an 86-kDa outer membrane receptor protein (PupA) which appears to be specific for ferric pseudobactin 358. PupA shares strong similarity with TonB-dependent receptor proteins ofEscherichia coli, which suggests a TonB-like protein in strain WCS358 is required for iron(III) transport. Strain WCS358 possesses a second uptake system for ferric pseudobactin 358 and structurally diverse ferric siderophores produced by other microorganisms. A second receptor gene (pupB) responsible for iron transport from pseudobactin BN7 or pseudobactin BN8 has been identified. The production of this and certain other ferric siderophore receptor proteins requires that strain WCS358 be grown in the presence of these siderophores. An apparent regulatory gene required for the expression ofpupB is located adjacent topupB. Two positive regulatory genes have been identified which can independently activate, under low-iron(III) conditions, transcription of genes coding for the biosynthesis of pseudobactin 358.
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  • 3
    ISSN: 1437-160X
    Keywords: Immunoglobulin allotypes ; Systemic lupus erythematosus ; Genetics ; Gm ; Km ; HLA-antigens ; Autoantibodies ; Clinical symptoms
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Immunoglobulin heavy chain (G1m, G2m, G3m, A2m) and kappa light chain (Km) allotype and phenotype frequencies of 323 central European Caucasian patients with systemic lupus erythematosus (SLE) were examined and correlated with various genetic, serologic and clinical markers of SLE. No significant associations were found between immunoglobulin allotypes or phenotypes and all 20 parameters tested (nephritis, vasculitis, arthralgias, photosensitivity, discoid lesions, central nervous system disease, Raynaud's phenomenon, sex, anti-Ro, anti-La, anti-nRNP, HLA-DR1-DR7, HLA phenotypes B8-DR3, B7-DR2). It could therefore be assumed that Gm, A2m and Km allotypes were not associated with HLA-antigens and had no influence on the serologic and clinical expression of SLE.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Annals of hematology 62 (1991), S. 188-189 
    ISSN: 1432-0584
    Keywords: Hemochromatosis ; Pyruvate kinase deficiency ; Hereditary anemia ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Hemochromatosis has been reported in several patients with chronic hemolytic anemia due to pyruvate kinase deficiency. We describe here a further patient with such an association and review the literature on the subject. We hypothesize that iron overload may occur in patients with pyruvate kinase deficiency who are also carriers of the hereditary hemochromatosis gene.
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  • 5
    ISSN: 1432-0428
    Keywords: Genetics ; diabetes mellitus ; restriction fragment length polymorphism ; glucose-transport ; familial
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Patients with Type 2 (non-insulin-dependent) diabetes mellitus and a strong family history of the disease may represent a sub-group where genetic factors play a pree-minent role in transmission of the disease. A defect in the liver/islet cell glucose transporter (GluT 2) could explain many of the pathophysiological features of the disease. In order to test the hypothesis that genetic variation at the GluT 2 locus contributes genetic susceptibility to Type 2 diabetes, 60 unrelated Caucasian diabetic patients with at least one affected sibling were genotyped for a Taq 1 restriction fragment length polymorphism marker. Hybridisation with a cDNA GluT 2 probe identified two alleles of sizes 13 kilobase (T1) and 19 kilobase (T2). The allele frequencies in the diabetic group with a family history were significantly different from those in a racially-matched control population of 122 subjects with no personal or family history of the disease (diabetic patients T1=0.96, T2=0.04, control subjects T1=0.89, T2=0.11, p〈 0.03). However, when the study was repeated with 54 diabetic patients with indeterminate family history, statistical significance was not reached although the allele frequencies showed a similar trend. The findings of this study support the hypothesis that a genetic variant of the liver/islet cell glucose transporter may contribute to familial susceptibility in Type 2 diabetes.
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  • 6
    ISSN: 1432-0428
    Keywords: Genetics ; Type 1 (insulin-dependent) diabetes mellitus ; Type 2 (non-insulin-dependent) diabetes mellitus ; HLA ; haptoglobin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Epidemiologic data suggest that having a parent with Type 2 (non-insulin-dependent) diabetes mellitus increases the risk for Type 1 (insulin-dependent) diabetes in siblings of a Type 1 diabetes proband. This increase in risk is consistent with a shared genetic susceptibility between Type 1 diabetes and Type 2 diabetes. We contrast genetic risk factors in three sets of families, consisting of (1) a single Type 1 diabetic child (proband) and non-diabetic parents, (2) multiple Type 1 diabetic siblings and non-diabetic parents, and (3) at least one Type 1 diabetic child and at least one Type 2 diabetic parent. Previous studies have demonstrated that HLA region genes, which elevate the risk in Type 1 diabetes, have no significant effect with respect to the risk for developing Type 2 diabetes. An earlier report cited a contribution by the haptoglobin locus to genetic susceptibility for Type 2 diabetes. We provide evidence that a high risk HLA antigen (HLA-DR3) is decreased to a greater extent in Type 1 patients with a Type 2 parent than in Type 1 patients in which the parents are not diabetic. The role of HLA-DR4 is maintained in these families, with an unexpectedly significant increased rate of transmission of the HLA-DR4 allele from Type 2 parent to Type 1 offspring. The role of haptoglobin in these families does not appear to be important, either with respect to association with diabetes or with respect to linkage with a secondary susceptibility locus. These results indicate that families with a Type 2 parent and Type 1 child, heavily determined by HLA-DR4 linked factors, may represent a homogeneous subset of diabetes susceptibility.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Oecologia 86 (1991), S. 243-250 
    ISSN: 1432-1939
    Keywords: Daphnia ; Life-history ; Genetics ; Variation ; Maturation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Life-history traits of 101 clones from two populations of Daphnia magna were measured under controlled environmental conditions in the laboratory. Some individuals had four juvenile instars, others had five. This depended on their length at birth and on the population they came from. Females in the group with five juvenile instars were smaller at birth but larger and older at maturity than those with four juvenile instars. Within groups of females with equal numbers of preadult instars (instar groups) age and size at maturity increased with size at birth. This relationship differed significantly among instar groups for both age and size at maturity. Significant differences in age and size at maturity between two populations became non-significant when size at birth was used as a covariable in AN-COVA. Within populations, size at birth depended on the clone and on the parity of the clutch. First-clutch offspring were considerably smaller than those from later clutches. The results suggest that variability in life-history traits is common within and between clones, but that most of this variation can be accounted for by size at birth and the number of pre-adult instars.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Psychopharmacology 104 (1991), S. 17-21 
    ISSN: 1432-2072
    Keywords: MK-801 ; Phencyclidine ; Ketamine ; CGP 39551 ; CGS 19755 ; NPC 12626 ; Locomotor activity ; Genetics ; NMDA/glutamate receptor complex ; Mice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of non-competitive (MK-801, phencyclidine, and ketamine) and competitive (CGP 39551, CGS 19755, and NPC 12626) N-methyl-d-aspartate (NMDA) receptor antagonists on locomotor activity in inbred CBA and C57, and in outbred NMRI mice were examined. Administration of the non-competitive NMDA antagonists produced a dose-dependent increase in well-coordinated locomotor activity at lower doses, followed by a bizarre behavioral syndrome (head weaving, body rolling, rotations, ataxia) after higher doses. The pharmacological profile of the competitive antagonists CGP 39551, CGS 19755, and NPC 12626 was more complex. CGP 39551 dose-dependently inhibited locomotor activity, whereas CGS 19755 and NPC 12626 displayed a biphasic action, that is low doses inhibited locomotor activity, whereas higher doses produced mild behavioral stimulation. The behavioral effects of NMDA antagonists appear to be genetically determined, since CBA animals were most sensitive to both noncompetitive and competitive antagonists, followed by NMRI and C57 animals. The differential effects of NMDA antagonists in various strains of mice suggest that the observed behavioral differences may be due to genetic differences in the NMDA/glutamate receptor channel complex.
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  • 9
    ISSN: 1432-072X
    Keywords: Carboxydotrophic bacteria ; Ribulosebis-phosphate carboxylase ; Phosphoribulokinase ; Hybridization ; Plasmids ; Genetics ; CO2 fixation ; Alcaligenes eutrophus ; Pseudomonas carboxydovorans ; Rhodospirillum rubrum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Heterologous gene probes derived from cfxLp and cfxPp genes of Alcaligenes eutrophus H16 revealed the presence of structural genes encoding ribulosebisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) on the genome of carboxydotrophic bacteria. The two genes were found to be rather conserved. In Pseudomonas carboxydovorans OM5 cfx genes reside on the plasmid pHCG3 and the chromosome as well, indicating that they are duplicated. Also in all plasmidharboring carboxydotrophic bacteria cfxL and cfxP structural genes were found to be plasmid-coded. Our results extend the list of carboxydotrophy structural genes residing on the plasmid pHCG3 and strongly support the idea that the components essential for the chemolithoautotrophic utilization of CO by Pseudomonas carboxydovorans OM5 are plasmid-coded. A cfxL gene probe from Rhodospirillum rubrum did not detectably hybridize with DNA from any of the carboxydotrophic bacteria examined.
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  • 10
    ISSN: 1432-2072
    Keywords: Habituation ; GABA ; Ethanol sensitivity ; Ethanol tolerance ; Genetics ; Mice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Habituation to a test environment following daily exposure for 5 days was examined in three genetically different strains of mice. C57 animals showed significant habituation to the new environment already on the second day. The habituation of NMRI mice was significant on the third day, whereas CBA mice showed no habituation at all during the experimental period. There was no difference between the animal strains in learning capacity in a passive avoidance test, but CBA mice displayed a significant increase in latency in their performance. When tested for sensitivity to the convulsant actions of GABAergic antagonists, picrotoxin produced seizures at lower doses in CBA as compared to NMRI and C57 mice, whereas there was no difference between the strains in the seizure activity produced by the specific GABA receptor antagonist bicuculline. When the animals were tested for sensitivity to ethanol in a horizontal wire test, ethanol (2 g/kg, IP) produced muscle relaxation in CBA mice whereas the performance of NMRI and C57 was not affected. A large dose of ethanol (4 g/kg, IP) produced a significantly longer sleeping time in CBA mice as compared to NMRI and C57 animals. Ethanol-produced hypothermia was, however, similar in all animals. Environment-dependent development of tolerance to ethanol following daily injections of ethanol for 4 days was examined. C57 mice showed the most rapid development of tolerance towards ethanol's hypnotic actions, whereas CBA mice showed no tolerance to this effect of ethanol. No difference between the strains to the development of tolerance to ethanol's hypothermic effects was observed. The present findings indicate that sensitivity to ethanol and ethanol tolerance are complex phenomena which cannot be adequately characterized by measuring only one single functional response to ethanol. The possibility that a genetically determined perturbation in the functions of the GABA receptor-coupled chloride channel, noted as variable sensitivity to picrotoxin, may be of importance for the observed disturbance in habituation to a new environment, for the different sensitivity to ethanol, and for the different rate of development of ethanol tolerance is discussed.
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    European archives of psychiatry and clinical neuroscience 240 (1991), S. 188-190 
    ISSN: 1433-8491
    Keywords: Families ; Genetics ; Polydiagnostic approach ; Schizophrenia ; Swedish family complex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A polydiagnostic computerized diagnostic system for psychosis was used in a Swedish family complex, and 51 patients with psychiatric symptomatology were examined with eight main diagnostic systems for schizophrenia and three systems for schizophrenic subgroups. All patients fulfilled the criteria for schizophrenia according to Taylor et al., 50 according to Carpenter, 41 according to RDC, and 31 of the 51 according to DSM-III and DSM-III-R. The hypothesis that the patients in the Swedish family complex differ from other phenotypes of schizophrenia must be refuted based on the data of the present study.
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 83 (1991), S. 24-32 
    ISSN: 1432-2242
    Keywords: Genetics ; Growth curve ; Body weight ; Chickens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Genetic improvement in growth of poultry has traditionally proceeded via selection for body weight at a fixed age. Due to increased maintenance costs and reproductive problems of adult broiler breeders, the potential for genetic manipulation of the growth curve has been receiving increased interest. Research of both male and female progeny of a three-way diallel cross was used to investigate the inheritance of growth curve parameters. The Laird form of the Gompertz equation was used to determine growth curve parameters, and was suited to the juvenile growth data frequently collected from meat-type chickens. Growth rate exhibited significant heterosis due to both autosomes and the sex chromosomes. Age at inflection point also exhibited significant average heterosis, though only among females. Growth rate was also influenced by average line effects, as was age at inflection point. Maternal effects had no influence on growth curve parameters, while additive sex linkage was observed for growth rate. Phenotypic and genetic correlations were calculated among the growth curve parameters and suggest that specific breeding programs could alter the growth trajectory of the contemporary broiler chicken. Moderate heritabilities were observed for the growth curve parameters and support the hypothesis that the growth curve could be altered via genetic manipulation of early postnatal growth, especially during the first 14 days post-hatch.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 82 (1991), S. 761-764 
    ISSN: 1432-2242
    Keywords: Rye ; Male sterility ; Genetics ; Gene location ; Trisomies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The genetics and relationships between the genes in rye located in the nucleus and cytoplasm of the male sterility of the G-type were investigated. A factor inducing male sterility was found in the cytoplasms or rye cv Schlägler alt and rye cv Norddeutscher Champagner. Monogenic inheritance was observed in linkage tests. Using primary trisomies of rye cv Esto, the nuclear gene ms1 was found to be located on chromosome 4R. Modifying genes, probably masked in normal cytoplasm but expressed in male-sterility-inducing cytoplasm together with gene ms1, were located on chromosomes 3R (ms2) and 6R (ms3). Mono-, di-, and trigenic inheritance types were found in backcross progenies of trisomies.
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 81 (1991), S. 50-58 
    ISSN: 1432-2242
    Keywords: Vicia faba ; Legumin ; Vicilin ; Structure ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different α-major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1α, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 82 (1991), S. 771-776 
    ISSN: 1432-2242
    Keywords: Secale cereale ; RFLP ; α-Amylase ; Genetics ; Isozymes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Rye α-Amy1, α-Amy2, and α-Amy3 genes were studied in the cross between inbred lines using wheat α-amylase cDNA probes. The α-Amy1 and α-Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the α-Amy3 region was much more conserved. The numbers of restriction fragments found and the F2 segregation data suggest that there are three α-Amy1 genes, two or three α-Amy2 genes, and three α-Amy3 genes in rye. These conclusions were supported by a simultaneous study of α-amylase isozyme polymorphism. The F2 data showed the three individual α-Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two α-Amy2 genes were shown to be separated by 5 cM. Linkage data within α-Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes.
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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    European journal of epidemiology 7 (1991), S. 490-493 
    ISSN: 1573-7284
    Keywords: Creutzfeldt-Jakob diseases ; Prion disease ; Jews ; Libya ; Genetics ; Pathophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The focus of CJD among Jews of Libyan origin has been recognized for two decades, but the reasons underlying it were unknown. Prevailing views suggested transmission from sheep infected with scrapie. However, recent data show that in fact CJD in this ethnic group is a genetically determined disease due to a point mutation on the codon 200 of the prion protein gene. The clinical characteristics of CJD in this group, and particularly the less common periodic activity in the EEG, are reviewed. New findings include peripheral neuropathy of the demyelinating type in two cases, presumably due to involvement of Schwann cells. The pathophysiology of the disease includes, presumably, a focal post-translational modification of the prion protein, (predisposed by the mutation). Later, the disease progresses through cell-to-cell transmission.
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 228 (1991), S. 361-371 
    ISSN: 1617-4623
    Keywords: Plant ; Hormone ; Genetics ; Hypocotyl ; Development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have isolated nine independent auxin-resistant mutants of Nicotiana plumbaginifolia by culturing M2 seedlings in the presence of indole-3-acetic acid ethyl ester or 1-naphthaleneacetic acid at concentrations which significantly inhibit hypocotyl elongation of the wild type. The mutations were induced by treating seed with ethyl methanesulphonate and were found in the course of screening 10 000 individual M2 families. Auxin resistance was in all cases the result of a mutation at a single, nuclear locus. The dominance relationships of two of the mutants could be defined as recessive or dominant; all other mutants showed partial dominance. In contrast to previously described mutants of Arabidopsis and N. plumbaginifolia, all of the present mutants were specifically resistant to auxin; the mutants were cross-resistant to several auxins, but showed no increased resistance to cytokinin, abscisic acid, ethylene or 1-amino-cyclopropane-1-carboxylic acid. The importance of the choice of the selection criterion for the isolation of specific resistance traits is discussed.
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  • 18
    ISSN: 1573-6903
    Keywords: Genetics ; catecholamine ; brain ; imprinting ; development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract This study was designed to compare catecholamine concentrations among three brain areas of four pureline populations of visually isolated chicks. The purelines used were a commercial male line, a fertility selected line, an unselected fertility control line, and unselected White Jersey Giants. In general, male chicks had significantly larger brain weights than females. Six catecholamine-related compounds (norepinephrine, epinephrine,l-DOPA, dopamine, DOPAC, and MHPG) were measured via HPLC-ECD. No significant differences in neurochemical concentration were observed for any line or brain area due to sex of the chick. The hypothalamus (HT) contained the greatest concentration of catecholamines in all lines, followed by the intramedial hyperstriatum ventrale (IMHV) and optic tectum (OT). The HT exhibited consistent lateralization in all lines with the right HT containing ca. 30% more catecholamines than the left HT. While no consistent lateralization was observed among the other brain areas, the IMHV exhibited significantly different degrees of lateralization among the populations. Neuronal activity, as measured by MHPG:NE and DOPAC:DA ratio varied by line within each brain area. There were line differences for MHPG:NE in the HT, IMHV, and OT, while line differences for DOPAC:DA were observed in the HT. Since differences among purelines have been demonstrated in this study, care must be given to precisely define the genotype of chicks used in behavioral and neurochemical research.
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  • 19
    ISSN: 1573-5060
    Keywords: Brassica oleracea ; Cauliflower ; Stalk rot ; Screening ; Genetics ; Resistance ; Sclerotinia sclerotiorum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The inheritance of resistance in cauliflower to stalk rot (Sclerotinia sclerotiorum (Lib.) de Bary) was investigated in population from six generations of six crosses. Disease incidence was recorded on 4 parents, 6 Fs 1, 6 Fs 2 and 12 back-crosses in a screenhouse under artificially created epiphytotic conditions. Resistance to stalk rot in this set of parents was found to be polygenic and under the control of recessive genes and due primarily to additive gene action. A breeding strategy emphasizing recurrent selection should lead to improvement in resistance.
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  • 20
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 12 (1991), S. 272-280 
    ISSN: 0192-253X
    Keywords: Cell differentiation ; cyclic AMP ; spore viability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sporogenous mutants of the cellular slime mold Dictyostelium discoideum are defined as mutants which are able to undergo terminal differentiation into spores in monolayer cultures in the presence of millimolar amounts of exogenous cyclic AMP. We describe the morphological development and cellular differentiation of a collection of 12 independently isolated sporogenous mutants of strain V12 M2. All mutants develop more rapidly than do wild-type at an air-water interface, display aberrant morphogenesis, and show overt spore and stalk differentiation as soon as 4 hr after starvation. All mutants differentiate in submerged monolayer culture in the presence of cAMP into variable proportions of spores and stalk cells. A number of the mutants also form both stalk cells and spores in submerged culture in the absence of exogenous cAMP. The spores formed by many of the mutants have a greatly reduced viability. Using parasexual genetics, we have found that two of the 12 mutants analyzed are dominant to wild-type and the remaining ten fall into a minimum of four complementation groups, the overall analysis thus yielding a minimum of four and a maximum of seven complementation groups. Intracellular cAMP levels in vegetative cells are significantly elevated in the two dominant mutants but are similar to wild type in all the other mutants.
    Additional Material: 3 Ill.
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  • 21
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 12 (1991), S. 293-298 
    ISSN: 0192-253X
    Keywords: Neural development ; messenger RNA ; somatostatin ; glial fibrillary acid protein ; proteolipid protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To examine the effects of ethanol exposure on neural development, pregnant rats were fed a liquid diet in which 37.5% of the total caloric content was ethanol-derived. The developmental appearance of the messenger RNAs coding for preprosomatostatin, glial fibrillary acidic protein, and proteolipid protein was examined by Northern blotting of total cellular RNA obtained from forebrain and hindbrain at various times after birth. In general, there was a delay in the developmental pattern of appearance of these mRNAs which was most noticeable at the early postnatal times. These results suggest that the previously reported delay in neural maturation is reflected at the level of the gene expression.
    Additional Material: 2 Ill.
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  • 22
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 12 (1991), S. 281-292 
    ISSN: 0192-253X
    Keywords: Actin function ; cell wall ; cytoskeleton ; fusion proteins ; mating ; 10 nm filaments ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of homologous proteins that are not closely related to other known proteins [Haarer BK, Ketcham SR, Ford SK, Ashcroft DJ, and Pringle JR (submitted)]. Temperature-sensitive mutants defective in any of these four genes display essentially identical pleiotropic phenotypes that include abnormal cell-wall deposition and bud growth, an inability to complete cytokinesis, and a failure to form the ring of 10 nm filaments that normally lies directly subjacent to the plasma membrane in the neck region of budding cells. We showed previously that the CDC3 and CDC12 gene products localize to the region of the mother-bud neck and are probably constituents of the ring of 10 nm filaments. We now report the generation of polyclonal antibodies specific for the CDC11 product (Cdc11p) and the use of these antibodies in immunofluorescence experiments with wild-type and mutant cells. The results suggest that Cdc11p is also a constituent of the filament ring, and thus support the hypothesis that the S. cerevisiae 10 nm filaments represent a novel type of eukaryotic cytoskeletal element. Cdc11p and actin both localize to the budding site well in advance of bud emergence and at approximately the same time, and both proteins also remain localized at the old budding site for some time after cytokinesis. Cdc11p also localizes to regions of cell-wall reorganization in mating cells and in cells responding to purified mating pheromone. Surprisingly, most preparations of affinity purified Cdc11p-specific antibodies also stained the nuclear and cytoplasmic microtubules. Although this staining probably reflects the existence of an epitope shared by Cdc11p and some microtubule-associated protein, the possibility that a fraction of the Cdc11 p is associated with the microtubules could not be eliminated.
    Additional Material: 4 Ill.
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  • 23
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 7 (1991), S. 1-14 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; cell cycle ; budding ; spore germination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cloning and sequencing of RCS1, a Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3′-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from α-factor treatment equally as well as wild-type cells.
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  • 24
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; alcohol oxidase ; amine oxidase ; choline ; peroxisome-deficient mutant ; enzyme assembly ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied the expression of alcohol oxidase (AO) in a peroxisome-deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose-limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild-type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3-dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol-grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.
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  • 25
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 26
    ISSN: 0749-503X
    Keywords: Spontaneous mutagenesis ; repression and derepression of genes ; environmental effects ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Spontaneous mutation of some genes was studied in haploid adenine and leucine auxotrophic yeast Saccharomyces.It was shown that a decrease in the amount of adenine (from 500 to 0 mgl-1) or leucine (from 300 to 0·3 mgl-1) in the medium, simultaneously with the transition from repression to derepression of the biosynthesis of these metabolites, resulted in a 15- to 150-fold increase in the reversion rate of genes ade2 and leu2, respectively, for different strains. At the same time the mutation rate of suppressor genes varied relatively little (up to five-fold), and that of gene lys1 did not change at all. It was also demonstrated (on gene leu2) that the mutation rate is determined by the composition of the nutrient medium at the time of the S-phase of the cell cycle and it does not depend on the cultivation conditions during the presynthetic period.We discuss the hypothesis that derepressed genes mutate with a significantly higher rate than genes in the repressed state.
    Additional Material: 5 Ill.
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  • 27
    ISSN: 0749-503X
    Keywords: Cytochrome c peroxidase ; hydrogen peroxide ; energetics ; yeast ; anaerobic respiration ; chemostat ; mitochondria ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Chemostat cultures of a catalase-negative mutant of Hansenula polymorpha CBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2 degradation is probably the mitochondrial enzyme cytochrome c peroxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochrome c as an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2 degradation by CCP must be provided by the respiratory chain.That H2O2 can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2 in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate.Energetic aspects of mitochondrial H2O2 decomposition via CCP and the physiological function of CCP in yeasts are discussed.
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  • 28
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    Yeast 7 (1991), S. 157-165 
    ISSN: 0749-503X
    Keywords: Translation initiation ; codon usage: mRNA structure ; yeast ; lacZ fusion protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A set of 32 different codons were introduced in a lacZ experssion vector (pPTK400) immediately 3′ from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of β-galactosidase fusion protein using immuno-gel electrophoresis. A 5·3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinatnts of the optimal context for an initiation codon in yeast therfore must be located mainly 5′ from this codon.
    Additional Material: 1 Ill.
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  • 29
    ISSN: 0749-503X
    Keywords: Translation ; Saccharomyces cerevisiae ; lacZ fusion ; termination ; nonsense suppression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons, UAA, UAG and UGA in Saccharomyces cerevisiae. The assay employs a 3-phosphoglycerate kinase-β-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is distupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring β-galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi-] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [pse+] broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA 〉 UAG 〉 UGA.
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  • 30
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    Yeast 7 (1991), S. 219-228 
    ISSN: 0749-503X
    Keywords: Protein kinase ; Saccharomyces cerevisiae ; yeast ; protein phosphorylation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145 000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as detrmined by the transfer of label from [γ-32P]ATP to either pp145KIN1 or to an exogenously added substrate, α-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in detrmining the role of phosphorylation in cellular growth and physiology.
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  • 31
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    Yeast 7 (1991), S. 265-273 
    ISSN: 0749-503X
    Keywords: cell division cycle ; Saccharomyces cerevisiae ; CDC 15 ; protein kinase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cell division cycle gene CDC 15 is essential for the late nucler division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC 15 gene product, as deduced from the nucletide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/theonine kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth are known. CDC 15 represents an essential protein kinase like CDC 7 and CDC 28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.
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  • 32
    ISSN: 0749-503X
    Keywords: Protein dislfide isomerase (PDI) ; chromosome III ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have determined the nucleotide sequence of the YCL313 gene as part of the YIp5 A1G clone localized on the left arm of chromosome III. This YCL313 gene encodes a protein of 522 amino acids (MW 58·3 kDa) which has large homologies with the human, mouse, chicken, bovine and rat PDI gene products. In these organisms the PDI gene encodes the protein disulfide isomerase (EC 5.3.4.1) also called S-S rearrangase, an enzyme that catalyses the rearrangements of S-S bonds in proteins. This enzyme is probably involved in protein folding within the lumen of the endoplasmic reticulum.These sequence homologies suggest that YCL313 is the yeast equivalent of the PDI gene. Gene disruption of YCL313 leads to a lethal phenotype indicating that this gene is essential for cell survival.
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  • 33
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; transcripts ; genetic distances ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A physical map of a contiguous DNA fragment of 60 kb, extending from the centromere to TRP5 on the left arm of the chromosome VII of Saccharomyces cerevisiae, strain IL 125-2B, was established. Within a 31 kb region from PMA1 towards TRP5, a total of 12 transcription products ranging from 0·6 to 3·6 kb were identified in cells grown exponentially on rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2·3 kb average length, was detected every 2·7 kb of DNA. The physical and genetical distances between the markers CEN7, pma1, leu1, pdr1 and trp5 were compared. A recombination frequency of 1 cM corresponds to an average distance of 3·3 kb between alleles in this region of chromosome VII.
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  • 34
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    Yeast 7 (1991), S. 305-308 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 1 Ill.
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  • 35
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 7 (1991), S. 313-323 
    ISSN: 0749-503X
    Keywords: Yeasts ; cell walls ; porosity ; proteins ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 2 Ill.
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  • 36
    ISSN: 0749-503X
    Keywords: Yeast ; Chromosome III ; sequence ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have entirely sequenced YCR59, which is a 10·1 kb segment of the right arm of chromosome III, and is a part of the clone E5F from the Newlon collection. The segment contains two long open reading frames (ORFs): YCR591 which starts in the adjacent fragment H9G (situated towards CRY1 and the centromere), and continues with 1833 codons in YCR59. The second ORF YCR592 is 1226 codons long and encoded entirely within YCR59. The two ORFs represent 91% of the total length of the segment. Excellent agreement in both location and length is found between the ORFs YCR591 and YCR592 and the transcripts 86 and 87 respectively in the Yoshikawa and Isono (1990) map of chromosome III. The two ORFs correspond to new genes and show no significant similarity with any known genes.
    Additional Material: 2 Ill.
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  • 37
    ISSN: 0749-503X
    Keywords: Secretion ; methylotrophic yeast ; glycosylation ; methanol oxidase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the α-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 μm plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the α-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the α-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active α-galactosidase enzyme was efficiently secreted (〉85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified α-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted α-galactosidase was glycosylated and had a sugar content of 9·5%. The specific activity of the α-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for guar α-galactosidase. Deglycosylation of the H. polymorpha α-galactosidase restored the specific activity completely.
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  • 38
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; yeast ; unsaturated fatty acids ; phosphatidylinositol ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae was grown anaerobically in media supplemented with myritoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), γ-linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid. Cells from exponential-phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidycholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids. The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid. Analysis of the fatty-acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation. The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is discussed.
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  • 39
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 40
    ISSN: 0749-503X
    Keywords: Trichosporon cutaneum ; auxotrophic mutants ; UV-mutagenesis ; transformation of spheroplasts ; sib-selection ; integration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully in spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per μg of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementing cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed. T. cutaneum spheroplasts with an efficiency of 50-200 colonies per μg of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.
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  • 41
    ISSN: 0749-503X
    Keywords: Chromosome III ; sequencing ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non-essential gene, however it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.
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  • 42
    ISSN: 0749-503X
    Keywords: Chromosome III ; sequencing ; gene distruption ; kinase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 2 Ill.
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  • 43
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    Yeast 7 (1991), S. 693-698 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; protein sorting ; post-translational modification ; allantoin pathway ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The DAL3 gene has been sequenced and found to encode a 195 amino acid protein with a molecular weight of 21 727. The four carboxy-terminal amino acids of DAL3 product (Cys-Ile-Ile-Ile) are homologous to those (CAAX) previously shown to be the primary structural signal for post-translational farnesylation of yeast RAS protein and mating factor. This modification is reported to be responsible for membrane localization of proteins containing it. The upstream region of DAL3 contains six copies of a sequence that is homologous to the positively acting DAL UASNTR reported to be required for transcriptional activation of the DAL5 and DAL7 genes. Missing from the DAL3 upstream region were any sequences related to those shown to be required for a DAL7 response to inducer, the UIS element. This correlates with the previous report that DAL3 expression is independent of the allantoin pathway inducer.
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  • 44
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    Yeast 7 (1991), S. 479-487 
    ISSN: 0749-503X
    Keywords: Pyruvate decarboxylase ; yeast ; Candida utilis ; Kluyver effect ; glycosidase ; β-glucosidase ; anaerobic sugar fermentation ; aerobiosis ; anaerobiosis ; activation ; deactivation ; catabolite repression ; enzyme induction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The glucose-fermenting yeast, Candida utilis cannot use the β-D-glucoside, cellobiose, anaerobically, although it is able to do so aerobically. β-Glucoside transport and hydrolysis and pyruvate decarboxylase activities of this yeast were measured aerobically and anaerobically. β-Glucoside transport was five-fold faster aerobically than anaerobically, but there was no corresponding difference in β-glucosidase activity. Pyruvate decarboxylase activity varied greatly, being synthesized de novo in response to the presence of D-glucose and anaerobic conditions and about 50% deactivated on the removal of D-glucose or the addition of air. Activation and deactivation were rapidly reversible. Failure to utilize cellobiose anaerobically, in particular, and the Kluyver effect, in general, probably depends on much reduced glycolytic flux, associated under anaerobic conditions, with (i) lower transport rate, (ii) low substrate affinity of the relevant glycosidase and (iii) deactivation of pyruvate decarboxylase. So, in addition to the complex effects of oxygen, anaerobiosis and specific sugars on induction, repression and derepression, there are fine controls on pyruvate decarboxylase activity, leading to fast activation or deactivation of the enzyme.
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  • 45
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 3 Ill.
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  • 46
    ISSN: 0749-503X
    Keywords: chromatin ; yeast ; Schizosaccharomyces pombe ; ade6 gene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5′ half, ‘not positioned’ nucleosomes around the 3′ end and a nuclease-sensitive promoter region. Precisely positioned nucleosomes, but no nuclease-sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.
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  • 47
    ISSN: 0749-503X
    Keywords: Cell wall porosity ; cell cycle ; centrifugal elutriation ; synchronous growth ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cell of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-arrest. These cultures grew synchronously for at least two generations. The cell wall porosity incresed sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth. It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells.We examined the incorporation of mannoproteins into the wall during the cell cycle. SDS-extractable mannoproteins were incorporated continuously. However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease.
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  • 48
    ISSN: 0749-503X
    Keywords: Superoxide dismutase ; cytochrome P450 ; chromosome VIII ; Saccharomyces cerevisiae ; polymorphisms ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DNA sequencing and analysis of genomic DNA using the polymerase chain reaction were used to demonstrate that SOD1 and ERG11 are adjacent genes in Saccharomyces cerevisiae S288c and to establish the correct intergenic sequence of this segment on chromosome VIII.
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  • 49
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 50
    ISSN: 0749-503X
    Keywords: Chromosome III ; Saccharomyces cerevisiae ; mating type ; HML ; BUD5 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This paper reports the DNA sequence of a segment of 9·8 kb of the chromosome III. The sequenced DNA contains the MATα locus. The new sequence of the MATα locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MATα (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.
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  • 51
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    Yeast 7 (1991), S. 757-760 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome mapping ; nitrogen catabolic genes ; secretion genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 52
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    Yeast 7 (1991), S. 745-755 
    ISSN: 0749-503X
    Keywords: Fission yeast ; mating type expression ; silencer ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The fission yeast, Schizosaccharomyces pombe, expresses one of two alternative mating types. They are specified by one of two determinants (M or P) present at the mat1 locus. In addition, silent copies of M and P are present on the same chromosome. In the present work we demonstrate that the difference between the active and the silent stage of the P determinant is controlled by four repressive elements that are located at the silent locus. There are two elements to the left and two to the right of the mating type cassette. Both elements to the left and either one of the two elements to the right are required for an effective blockage of transcription. When they are combined, the four elements define a highly efficient silencer functionally similar to the HMRE and HMLE and HMLI silencers in Saccharomyces cerevisiae. In addition, the DNA surrounding the silent P locus confers symmetric partitioning in mitosis to Schizosaccharomyces pombe ars plasmids.
    Additional Material: 6 Ill.
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  • 53
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    Yeast 7 (1991), S. 775-780 
    ISSN: 0749-503X
    Keywords: Kluyveromyces ; lactic acid ; transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D-and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5·4: Vmax = 1·00 (±0·13) mmol h-1 per g dry weight and Ks = 0·42 (±0·08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simulatate uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.
    Additional Material: 6 Ill.
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  • 54
    ISSN: 0749-503X
    Keywords: Superoxide dismutase ; brewing yeast ; catalase ; oxygen toxicity ; aerobic transition ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The physiological effects on brewing yeast, growing in semi-defined wort medium, of a sudden transition from aerobiosis to anaerobiosis were studied. Two yeast strains were examined, used for ale and lager fermentations respectively. The reverse transition (from anaerobiosis to aerobiosis) was also examined. Transitions were applied by changing the sparging gas during growth or in stationary phase, and the effects on the specific activities of certain key enzymes and on the viability of the cultures were examined.Neither type of transition led to significant changes in growth rate, the rate of ethanol production or the specific activities of alcohol dehydrogenase and pyruvate decarboxylase. The most significant change was in the specific activity of CuZn-superoxide dismutase, which showed a rapid increase in activity on transition from anaerobiosis to aerobiosis, and a decrease in activity on the reverse transition. Catalase activity in the ale yeast generally followed that of CuZn-superoxide dismutase, whereas in the lager yeast it remained unchanged by the transitions. The transition from anaerobiosis to aerobiosis caused increases in citrate synthase and Mn-superoxide dismutase, though only after a significant lag period. Aerobic to anaerobic transitions caused a decrease in Mn-superoxide dismutase activity, while citrate synthase remained unchanged.Anaerobically grown cells showed a rapid loss in viability on exposure to oxygen (5-7% in the first hour), while aerobically grown cells were unaffected. When anaerobically grown cells were exposed to 0·25 mM-potassium superoxide, there was an 8% loss of viability within 10 min, whereas aerobic cells were not affected.It is concluded that the toxic effect of oxygen is due to superoxide (or a species derived from it) and that the CuZn-superoxide dismutase (but not the Mn-isoenzyme) plays a role in protecting the cells. The de novo synthesis of the CuZn-enzyme is not always rapid enough to confer full protection.
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  • 55
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    Yeast 7 (1991), S. 147-156 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; microsomes ; cytochrome P450 ; sterol demethylase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using cells grown in a chemostat at steady state, the levels of various components of the microsomal electron transport chain of Saccharomyces cerevisiae were examined. Cytochrome P450 haemoprotein levels measured in cells grown in medium with a dissolved oxygen concentration of 15% were induced between 10- and 20-fold over levels in cells grown in medium containing 70% dissolved oxygen concentation. An increase in the dilution rate of a culture growing in medium containing 15% dissolved oxygen resulted in an increase in the residual glucose concentration of the medium. This was paralleled by an increase in the microsomal levels of cytochrome P450. The rise could not be attributed either to increases in the concentration of ethanol in the chemostat or to an increase in the proportion of energy generated using fermentative pathways. However, this effect was not observed in cells grown in an oxygen concentration of 70%. Cytochrome b5 haemoprotein levels were also induced approximately three-fold by reducing the dissolved oxygen concentration from 70% to 15%. Changes in the medium glucose concentration from 0·03% to 1·6% (w/v) had no effect on the levels of this enzyme. Conversely, levels of cytochrome P450 NADPH reductase appeared lower in cells grown in 15% as opposed to 70% dissolved oxygen concentration. Northern slot blot analysis of total RNA extracted from chemostat-grown cells, probed with a C-14 sterol demethylase cytochrome P450 gene (cytochrome P450 LIA1), revealed a pattern of message induction which matched that of the cytochrome P450 haemoprotein, indicating that control of the levels of this enzyme was at least partially transcriptional. Qualitative examination of combined cytochrome P450 apoprotein and haemoprotein levels using Western blot analysis revealed a similar pattern of induction to that observed with Northern blotting.
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  • 56
    ISSN: 0749-503X
    Keywords: rDNA spacer probes ; rapid yeast identification ; Metschnikowia reukaufii ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7·7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0·65 kb BamHI-HpaI fragment containing nontranscribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclsively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G+C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.
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  • 57
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    Yeast 7 (1991), S. 195-209 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 11 Ill.
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  • 58
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    Yeast 7 (1991), S. 229-244 
    ISSN: 0749-503X
    Keywords: Lipid-binding proteins ; Saccharomyces cerevisiae ; secretion ; secretory vesicles ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast (Saccharomyces cerevisiae) cytosol was examined for the presence of calcium-dependent membrane- or lipidbinding proteins that might paly fundamental roles in membrane-associated phenomena in stimulated cells. A complex group of proteins was isolated from late log phase cultures of yeast strain YP3 on the basis of calcium-dependent association with yeast secretory vesicles isolated from the temperature-sensitive sec6-4 secretory mutant. The masses of the major proteins in this group were 32, 35, 47, 51, 55, 60, and 120 kDa. A similar group of proteins was isolated by calcium-dependent association with bovine brain lipids enriched in the predominant acidic phospholipids of the yeast secretory vesicles. The 47 kDa protein was highly purified when commerical yeast cake was used as the source of yeast cytosol. The 32 kDa and 60 kDa proteins were demonstrated to reassociate with lipids at calcium concentrations of 100 μM or higher, while no association was promoted by 2 mM-magnesium. The 47 kDa protein could be removed from lipids by reducing the calcium concentration to between 1 and 32 μM. The sequences of peptides isolated from digests of several of these proteins indicate that they are novel proteins but are insufficient to judge the possible homology of these proteins with mammalian membrane-binding proteins. The sequence data may be adequeate to permit isolation and modification of the corresponding genes in order to assess the possible funtion of this class of proteins in stimulated cells.
    Additional Material: 12 Ill.
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  • 59
    ISSN: 0749-503X
    Keywords: Cloning genes ; toxic ; E. coli ; non-selective transformation ; co-transformation ; one-step gene disruption ; deletion ; ligation libraries ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important need of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformations at a frequency of approximately 0·1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0·18%. Non-selective transformation using this transformation proctocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30-40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2-5% the frequency of the single event.
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  • 60
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; alcohol dehydrogenase ; mitochondrial import ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Suprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.
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  • 61
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 62
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    Yeast 7 (1991), S. 445-454 
    ISSN: 0749-503X
    Keywords: Heterologous expression ; S. cerevisiae ; α-glucosidase ; secretion ; maltose utilization ; novel promoter ; Candida tsukubaensis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The α-glucosidase gene of Candida tsukubaensis is contained within a 3·47 kb BamH1-Mlu1 fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast-Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S. cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other α-1,4-linked α-D-glucopyranosides, specifically hydrolysed by the α-glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70-80 kilodalton (kDa). Deglycosylation of the α-glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63-65 kDa (peptide 1) and 50-52 kDa (peptide 2). An increase in the level of expression of the α-glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2-d gene as selectable marker. The α-glucosidase gene promoter functions more effectively than the Gal1-10 promoter in directing α-glucosidase expression in S. cerevisiae. It also directs the expression of high levels of β-galactosidase activity in yeast when fused to a promoterless E. coli lacZ gene. Expression of the α-glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.
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  • 63
    ISSN: 0749-503X
    Keywords: RVS161 gene ; Saccharomyces cerevisiae ; stationary phase entry ; viability loss ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditons have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161-1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.
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  • 64
    ISSN: 0749-503X
    Keywords: Yeast ; vacuoles ; pyrophosphatase ; H+-transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The membrane-bound and soluble pyrophosphatase (PPase) activities of Saccharomyces carlsbergenis vacuoles are determined by the functioning of special enzymes and are not due to non-specific PPi hydrolysis by other vacuolar phosphohydrolases. The molecular mass of the membrane-bound PPase is apparently 120 000 and its molecule consists of three subunits with Mr = 41 000. Soluble PPase has a molecular mass of about 82 000 and includes three subunits with Mr = 28 000. Both enzymes are glycoproteins. The vacuolar membrane-bound PPase is a proton pump.
    Additional Material: 8 Ill.
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  • 65
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 66
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    Yeast 7 (1991), S. 717-726 
    ISSN: 0749-503X
    Keywords: Mannoprotein ; glucan ; mannan ; N-glycosylation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extrct of cell walls or in a hot-citrate extract of mnn9 cells.The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild β-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was β-glycosidically linked.The results indicate that β-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.
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  • 67
    ISSN: 0749-503X
    Keywords: Yeast ; Saccharomyces cerevisae ; endoplasmic reticulum ; ultrastructure ; cell cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double-labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.
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  • 68
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; plasmid stability ; plasmid gene expression ; ARS elements ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hydribization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.
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  • 69
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 70
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    Yeast 7 (1991), S. 37-50 
    ISSN: 0749-503X
    Keywords: Transposon Tn5 excision ; recombination ; LYS2 gene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Five complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature-sensitive mutation resulting in a 20-100-fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.
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  • 71
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    Yeast 7 (1991), S. 73-78 
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces ; fission yeast ; nDNA/nDNA optical reassociation ; taxonomy ; phylogenetic relatedness ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The genus Schizosaccharomyces comprises a small, somewhat heterogeneous group of yeast species which have in common a unique mode of vegetative reproduction by cross-wall formation without constriction as well as a certain degree of osmophilia, most strains having been isolated from habitats of high sugar concentration. This study evaluated inter- and intraspecific relationships utilizing nDNA/nDNA optical reassociation and by the analysis of physiological profiles of several strains of each species. Results demonstrate that the genus should be divided into three species: Schiz. japonicus, Schiz. octosporus and Schiz. pombe.
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  • 72
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    Yeast 7 (1991), S. 51-60 
    ISSN: 0749-503X
    Keywords: ARS elements ; plasmid ; mitochondrial DNA ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The insert-containing, non-canonical ori 6 region of yeast mitochrondrial DNA of Saccharomyces cerevisiae was dissected into 15 different segments that were ligated to the integrative yeast vector YIp5. Six recombinant plasmids exhibited replicative ability in yeast and carried consensus sequences similar to the previously described 11 bp motifs active as autonomous replication sequences (ARS). In addition, all active constructions carry one or more of the characteristic GC-rich domains A, B or C present in the ori 6 region, thus confirming and expanding the study of Blanc (Gene 30 (1984) 47-61) with the canonical ori 5. Also a new transcriptional origin is activated in the ori 6 region, apparently circumventing a disruption by insertion of a GC-rich sequence that, in this ori, removes the mitochondrial promoter usually present next to the Celement. The ARS-positive constructions correspond to the retained segments of spontaneous well-characterized suppressive or neutral petite genomes that contain segments of the ori sequence.
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  • 73
    ISSN: 0749-503X
    Keywords: Ascomycetous yeasts ; ribosomal RNA ; molecular evolution ; phylogeny ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Species of the genera Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces were compared from their extent of divergence in three regions from small (18S) and large (25S) subunit ribosomal RNAs comprising a total of 900 nucleotides. With the exception of the closely related Saccharomyces bayanus and S. pastorianus, which appear to have identical sequences, all other species could be distinguished by nucleotide differences in a variable region of the large subunit, and genus-specific nucleotides were discernible in all three regions. The taxon D. tamarii differed markedly from other species and is excluded from Debaryomyces. By contrast, Schwanniomyces occidentalis showed few nucleotide differences with Debaryomyces spp. and its transfer to Debaryomyces is proposed. Schizosaccharomyces proved to be somewhat more divergent than Saccharomyces and Debaryomyces, but species differences appear insufficient for dividing the genus. Some of the factors influencing estimates of phylogenetic distances from rRNA sequences are discussed.
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  • 74
    ISSN: 0749-503X
    Keywords: Nuclear proteins ; HMG proteins ; yeast ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to determine the biological functions of moderately abundant, high mobility group (HMG)-like nuclear proteins, a genetic approach has been taken. The gene for one such protein, NHP2, has been cloned and characterized from Saccharomyces cerevisiae. NHP2 has been called ‘HMG-like’ because of the physical/chemical properties it shares with the HMG proteins from higher eukaryotic cells. However, nucleotide sequence analysis revealed that NHP2 could encode a 17·1 kilodalton basic protein which was not significantly homologous to any previously sequenced HMG proteins. Thus NHP2 defines a new member of the HMG class of proteins. A search of protein databases showed that the amino acid sequence of NHP2 shared significant identities with two ribosomal proteins; the acidic ribosomal protein S6 from Halobacterium marismorium and protein L7a from mammals. The biological relevance of these homologies is nuclear since previous biochemical results indicated that NHP2 was not a ribosomal protein. S1 nuclease analysis indicated that the gene contained no introns but had multiple transcription initiation sites 20 to 40 bases before the ATG codon. Finally, NHP2 has been shown to have a critical role in the cell; when a diploid yeast strain deleted of one copy of the NHP2 gene was sporulated and dissected, only half of the spores grew into normal colonies. The rest of the spors germinated, but only formed microcolonies containing 12 to 40 cells. None of the spores which grew into normal-sized colonies contained the mutant NHP2 gene, thus demonstrating that the NHP2 protein has an essential physiological function.
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  • 75
    ISSN: 0749-503X
    Keywords: Metallothionein ; resistance to metal ions ; expression vectors ; CUP1 ; β-galactosidase ; upstream activation sequences ; recombinant DNA ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Shuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10·6 kb, have been constructed between the metallothioneinencoding CUP1 gene of Saccharomyces cerevisiae and a vector capable of replication in Kluyveromyces lactis. Introduction of these plasmids into K. lactis confers resistance to copper as well as to cadmium and silver. Resistance to these latter metal ions, in the absence of induction by copper, suggested that the CUP1 gene is constitutively expressed in the foreign background. Introduction of the lacZ reporter gene from Escherichia coli into a cloning site downstream from the CUP1 promoter showed that expression of this gene is constitutive in K. lactis but in S. cerevisiae induction by copper is necessary. Sequences upstream from the CUP1 promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance. It is suggested that a K. lactis protein, normally involved in activating transcription of the resident CUP1 gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introduced CUP1 gene.
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  • 76
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; yeast ; starch ; secretion ; extracellular glucoamylase ; feed-back control ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified: repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold difference in the rate of GA production) can be displayed by either glucose-limited or glycerol- plus ethanol-consuming cultures: the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated GA. It is proposed that the feed-back control involved in GA production can be attenuated by starch which can thus ‘induce’ higher rates of GA production compared to the basal mode.
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  • 77
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 78
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    Yeast 7 (1991), S. 211-217 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; cell wall mutant ; endocytosis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent an consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of hte dye at 37°C is 145 ng/μg DNA per h for VY1160 mutant and 23 ng/μg DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24°C and shifted at 38°C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38°C. The analysis of ts1 × wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might the generally increased permeability of VY1160 mutant cells.
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  • 79
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    Yeast 7 (1991), S. 245-252 
    ISSN: 0749-503X
    Keywords: K. lactis ; killer system ; ORF 10 ; DNA binding protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: ORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22·3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymeras/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA-Sepharose column, and is eluted by 350 mM-salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other proteins(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequeence homology between the ORF 10 protein and the family of histone-like proteins.
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  • 80
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 1 Ill.
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  • 81
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 1 Ill.
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  • 82
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    Yeast 7 (1991), S. 357-366 
    ISSN: 0749-503X
    Keywords: Fission yeast ; mating pheromone ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Although there have been several reports demonstrating the existence of mating factors in the fission yeast Schizosaccharomyces pombe it has not been possible to isolate these factors as cell-free preparations. Such an ability is the first requirement towards a molecular characterization of these factors and here I report the successful isolation of a mating factor from S. pombe. This factor, termed M-factor, is released by cells of the cellular mating type M (Minus) and induces mating-specific changes in P-type cells. A reliable and accurate assay for the quantitation of the M-factor, based upon changes in cell volume following exposure to the factor, is also described.
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  • 83
    ISSN: 0749-503X
    Keywords: Inositol ; biosynthesis ; yeast ; Candida albicans ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Candida albicans inositol biosynthetic gene and its regulation have been studied. The gene, CalNO1, was cloned on a multicopy vector by complementation of a Saccharomyces cerevisae mutant strain. Southern blot analysis established that the cloned DNA was C. albicans genomic DNA in origin; neither rearrangements nor pseudogenes were evident. Blot hybridization analysis using RNA isolated from C. albicans revealed that a single RNA species (1·8 kilobases) was homologous to the cloned DNA fragment. The steady-state levels of these transcripts were shown to be regulated in response to inositol in the growth media. In addition, the steady-state levels of the RNA encoded by the cloned C. albicans DNA present in S. cerevisiae on a plasmid (YRpCalNO1) were regulated in response to exogenously provided inositol. The cloned C. albicans DNA fragment was shown to restore inositol-1-phosphate synthase activity to a S. cerevisiae mutant strain defective in this enzyme. This activity was also shown to be regulated in response to the presence of inositol in the growth media.
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  • 84
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    Yeast 7 (1991), S. 337-346 
    ISSN: 0749-503X
    Keywords: Yeast ; ADHI promoter ; regulation ; heterologous expression ; increased production ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Production of Bacillus amyloliquefaciens α-amylase by Saccharomyces cerevisiae using the multicopy plasmid pAAH5 and ways of improving the yields of secreted enzyme were studied. In standard non-buffered medium, α-amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of α-amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the ADH1 promoter present on pAAH5 resulted in delayed but increased α-amylase production: 29-fold in selective medium, two-fold in non-selective medium. With the original ADH1 promoter, accumulation of α-amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with the appearance of a mRNA larger than the α-amylase messenger. With the shortened promoter, the normal-size α-amylase mRNA was detected under all growth conditions and α-amylase was efficiently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of α-amylase were obtained with the short promoter in non-selective glucose-containing medium; this may be explained by the greater final cell density obtained. However, the production of α-amylase per cell mass was higher in ethanol-containing selective medium. Seventy to eighty per cent of the α-amylase activity was secreted into the medium independent of the total amount produced.
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  • 85
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    Yeast 7 (1991) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 86
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    Yeast 7 (1991), S. 773-774 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 87
    ISSN: 0749-503X
    Keywords: Methionine/threonine biosynthesis ; transcriptional regulation ; general amino acid control ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genes encoding enzymes in the threonin/methionine biosynthetic pathwa were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
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  • 88
    ISSN: 0749-503X
    Keywords: Ribosomal RNA ; transcription ; enhancer ; promoter ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The promoter and enhancer of the rRNA gene of Saccharomyces cerevisiae have been studied using a nuclease S1 protection assay to detect transcripts of an rRNA minigene in transformed yeast. Analysis of 5′ deletion mutants showed that DNA between - 163 bp and - 155 bp was important for promoter activity and that some DNA between - 155 bp and - 145 bp was essential. The importance of DNA far upstream from the initiation site was confirmed by showing that minigene expression was much reduced by linker scanner mutations clustered around - 148 bp, - 133 bp and - 100 bp, and was abolished by mutations clustered around - 118 bp. The enhancer for rRNA biosynthesis increased transcription from all of the five mutated promoters that were tested. The magnitude of the enhancer effects on weakly active promoters was two- to three-fold less than on the wild-type promoter. Expression of a minor transcript in a 5′ deletion to - 10 bp was substantially reduced by a mutation which altered two base pairs in the core sequence of the promoter-proximal REB1 binding site.
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  • 89
    ISSN: 0749-503X
    Keywords: Glycogen phosphorylase ; phosphorylase monomers ; proteinase yscA ; proteolytic modification ; Saccharomyces ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Analyses by sodium dodecyl sulphate-polyacrylamide gradient-gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s-monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell. It is observed in extracts of Saccharomyces carlsbergensis and S. cerevisiae after incubation at ambient temperatures or even after storage in the frozen state at -25°C. Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants of S. cerevisiae that do not contain active proteinase yscA (EC 3.4.33.6). When purified proteinase yscA-deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc-monomer is formed in their extracts. This event must be attributed to a different proteinase, since it is sensitive towards p-hydroxymercuriphenylsulphonate, but not towards pepstatin A. The N-terminal amino acid of the sc-monomer was found to be blocked, as in the case of the native 1-monomer, but not the s-monomer.
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  • 90
    ISSN: 0749-503X
    Keywords: Kluyeromyces marxianus ; LEU2 gene ; β-isopropylmalate dehydrogenase ; leu2 mutant ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The LEU2 gene, coding for β-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5′-untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine-specific regulation of transcription. We show that the cloned gene can be used for the construction of a non-reverting K. marxianus leu2 mutant.
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  • 91
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    Yeast 7 (1991), S. 823-831 
    ISSN: 0749-503X
    Keywords: Yeast recombination ; RAD52-dependent recombination ; electroporation ; DNA damage ; induced recombination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae cells treated by high voltage and made transformation-competent (electroporation) are also made hyper-recombinational as determined by an assay that measures interchromosomal mitotic recombination between chromosome III homologs, each containing mutant heteroallelic copies of the trp1 and his3 genes. There is a 10-fold stimulation of Trp+ and 21-fold stimulation of His+ prototrophs. Although this stimulation coincides with conditions for maximal transformation competence it is independent of the presence of transforming plasmid DNA. Electroporation does not increase the reversion frequency of these mutations, nor is there a stimulation in Ty transposition. Among the electroporation-stimulated Trp+ and His+ recombinants there is no dramatic difference in the pattern of events: that is to say that, while there is an increase in the number of recombinants, the distribution of gene conversion and cross-over events among the stimulated recombinants is not significantly altered compared to spontaneously arising Trp+ and His+ recombinants. This electroporation-stimulated recombination is abolished in an isogenic rad52 mutant strain consistent with the increase in Trp+ and His+ prototrophs being the result of a stimulation of a RAD52-dependent recombination pathway.
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  • 92
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    Yeast 7 (1991), S. 849-855 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; amino acid transport ; Cysteine ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Although Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT(83·3 μM). The optimal pH of cysteine uptake was between 4·5 and 5·0, but the activity was quickly lost if cells were kept in buffer. When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride. Thioglycol as well as metabolic inhibitors such as dinitropherol and azide were inhibitory. Homocysteine and methionine were competitive and non-competitive inhibitors, respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH-group but also amino- and carboxyl-groups.In wild-type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source. On the other hand, in cysteine-dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine. From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains.
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  • 93
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    Yeast 7 (1991), S. 873-880 
    ISSN: 0749-503X
    Keywords: TDH2 ; MET3 ; methionine biosynthesis ; ATP sulphurylase ; glyceraldehyde-3-phosphate dehydrogenase ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al. Gene 34, 269-281, 1985) and nucleotide sequence (Cherest et al. Mol. Gen. Genet. 210, 307-313, 1987). Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C-based library had the same restriction map as the chromosomal DNA from both of the strains. Comarison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact. In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J. Biol. Chem. 255, 2596-2605, 1980). The chromosomal orientation of the two genes was determined to be MET3-TDH2-CEN10.
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  • 94
    ISSN: 0749-503X
    Keywords: 2 μm-plasmid stability ; copy number ; regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The 2 μm plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to high copy number and extreme stability. However, the cloning of genes into 2 μm often results in a reduced copy number and/or reduced stability. One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid. Therefore we studied the effect on stability of insertions in the silent regions of 2 μm without interrupting any known essential regions or ORFs. Using the SnaBI site, a yeast-integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations. The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild-type 2 μm plasmid. However, the stability of these plasmids in a cir° host was still one to two orders of magnitude lower (0·2% and 0·8% respectively) as reported for the wild-type 2 μm (0·01%). Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability. The stability was dependent on the orientation of the insert. We found that in the less stable orientation, transcription originating from the insert was running into the STB region.DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid. These effects are strongly dependent on the size of the insert. Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.
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  • 95
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    Yeast 7 (1991), S. 367-378 
    ISSN: 0749-503X
    Keywords: S. cerevisiae ; heat-shock proteins ; starvation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat-shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells.The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at vaious stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.
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  • 96
    ISSN: 0749-503X
    Keywords: DNA-binding proteins ; ARS ; centromere ; yeast ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: GFI and GFII are abundant DNA-binding proteins in the yeast Saccharomyces cerevisiae. Binding sites for GFI conform to the sequence 5′-RTCRYNNNNNACG-3′. This consensus can also accommodate the recognition sequence for the ARS1-binding factor ABFI. Results of retardation-competition assays, limited proteolysis experiments, molecular weight determinations based on denaturation-renaturation procedures and mobility shift assays of protein-DNA complexes formed in the presence of a monoclonal antibody raised against ABFI suggest strongly that GFI and ABFI are the same protein. Similarly, GFII appears to be identical to the centromere-binding protein CPFI (alias CP1), since both proteins bind to the CDEI motif of yeast centromeres (5′-RTCACRTG-3′) and cannot be detected in a cpf1 disruption mutant yeast strain. In addition, based on denaturation-renaturation studies, both factors appear to have molecular weights in the same range of 53-62 kDa.
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  • 97
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; expression system ; S and L surface antigens ; hepatitis B vaccine ; multimeric ; non-homologous integration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H. polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
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  • 98
    ISSN: 0749-503X
    Keywords: Plasmids ; gene disruption ; selectable cassettes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.
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  • 99
    ISSN: 0749-503X
    Keywords: Asporogenic yeast ; Candida tropicalis ; Peroxisomes ; POX genes ; pulsed-field gel electrophoresis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The peroxisomes of the asporogenic yeast Candida tropicalis contain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11 POX genes encoded independent PXPs and three POY genes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes of C. tropicalis were separated by pulsed-field gel electrophoresis. Eight chromosomal bands were observed over the range of 1·0 Mbp (band I) to 2·8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands: POX6A and POX8B, on bands V and VII; and POX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by either NotI or SfiI endonuclease. The results suggested that C. tropicalis was diploid and that restriction sites were conserved little between homologues. The three POX genes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal bands.
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  • 100
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    Yeast 7 (1991), S. 539-546 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; heat-shock element ; lacZ induction ; growth state ; ubi4, ubc4, ubc5, pep4-3 mutants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Heat-shock induction of heat-shock protein genes is due to a specific promoter element (the heat-shock element, HSE). This study used lacZ under HSE control (HSE-lacZ) to characterize HSE activity in Saccharomyces cerevisiae cells of different physiological states and differing genetic backgrounds.In batch fermentations HSE-lacZ induction by heat shock was maximal in exponential growth, and showed marked decline with the approach to stationary phase. Expression in the absence of heat shock was unaffected by growth phase, indicating that the growth-dependent expression of many yeast heat-shock genes uses promoter elements in addition to the HSE. Heat-induced expression was strongly influenced by the temperature at which cultures were grown. While basal, uninduced expression was constant during growth at different temperatures to 30°C, induction by transfer to 39°C was reduced by increases in growth temperature as low as 18-24°C. Maximal HSE-lacZ induction (30- to 50-fold) was in cultures grown at low temperatures (18-24°C), then heat shocked at 39°C. Ethanol was a poor inducer. Mutations having little effect on HSE-lacZ expression included a respiratory petite; ubi4 (which inactivates the polyubiquitin gene); also ubc4 and ubc5 (which each inactivate one of the ubiquitin ligases involved in degradation of aberrant protein). pep4-3 increased both basal and induced β-galactosidase about two-fold, probably because of slower turnover of this enzyme in pep4-3 strains.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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