Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000.

The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content.     

Immunochemical properties of mannan-protein complex isolated from viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain by the action of zymolyase

Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp. beta-1,3-glucanase, Zymolyase-60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca. 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135 C. The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S. cerevisiae X2180-1A wild and S. cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction. Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity.     

Synthesis of a mannose heptasaccharide existing in baker's yeast,Saccharomyces cerevisiae X2180-1A wild-type strain

A mannose heptasaccharide existing in baker's yeast, Saccharomyces cerevisiae X2180-1A wild-type strain, was effectively synthesized as its allyl glycoside via TMSOTf-promoted condensation of a disaccharide donor 13 with a pentasaccharide acceptor 12, followed by deprotection. The pentasaccharide 12 was constructed by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9) with allyl 6-O-acetyl-3,4-di-O-benzoyl-alpha-D-mannopyranoside (10), followed by deacetylation. The tetrasaccharide 9 was obtained by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5) with allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (6), followed by deallylation and trichloroacetimidation. The disaccharides 6 and 13 were readily obtained by known methods.     

A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element

In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences. They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome. These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat. In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.     

Relationship between phosphate content and serological activities of the mannans of Candida albicans strains NIH A-207, NIH B-792, and J-1012.

The mannans from Candida albicans strains NIH A-207 (serotype A), NIH B-792 (serotype B), and J-1012 (serotype C) were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions containing different amounts of phosphate. Antibody-precipitating activities of the mannan subfractions of strains NIH A-207 and NIH B-792 were proportional to their phosphate content, while those of strain J-1012 did not show regularly proportional precipitin activity. A similar tendency was also observed in the cross-reaction between the mannan su,fractions of strains NIH A-207 and J-1012 and their heterologous antisera. The mannans of strain NIH B-792 showed lower cross-reactivities against antisera of strains NIH A-207 and NIH B-792, i.e., only two subfractions containing larger amounts of phosphate were able to react with these antisera.     

DNA-repair characterization of cdc40-1, a cell-cycle mutant of Saccharomyces cerevisiae

The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation. cdc40-1 mutants shown only slight sensitivity to UV irradiation. Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS. rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures. An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature. UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.     

Gene dosage effects in polyploid strains of Saccharomyces cerevisiae containing gua-1 wild-type and mutant alleles.

Triploid and tetraploid Saccharomyces strains containing different combinations of a gua-1 mutant allele and the corresponding wild type were prepared. The cultivation of the different strains in media upon which the mutant fails to grow leads to a pronounced growth rate response to the dosage of the wild-type allele. Proportionality between the specific activity of the guanosine 5'-monophosphate synthetase and the wild-type dosage was reavealed. Inosine 5'-monophosphate dehydrogenase, the precursor enzyme in the pathway, is derepressed in a sigmoid manner when the wild-type dosage is reduced, whereas the activity of cytosine deaminase, investigated as a reference enzyme, is less affected.     

Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide

The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 mM HCl at 100 degrees C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of beta-1,2-linked D-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies.     

Elevated growth of Saccharomyces cerevisiae ATH1 null mutants on glucose is an artifact of nonmatching auxotrophies of mutant and reference strains

Yeast strains disrupted for ATH1, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the URA3 gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.     

Growth-inhibitory activity of the d-mannan of saccharomyces cerevisiae X2180-1A-5 mutant strain against mouse-implanted sarcoma 180 and ehrlich-carcinoma solid tumor

The d-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain, which possesses a main chain composed of α-(1→6) linked d-mannopyranosyl residues and a small proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, showed strong growth-inhibitory activity against mouse-implanted Sarcoma 180 and Ehrlich-carcinoma solid tumor. The observation that the level of this activity was nearly identical with that of the d-mannan of a wild-type strain of bakers' yeast, which possesses a high proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, suggests that the branches are not essential for antitumor activity. The partial acid-degradation products of both d-mannans, the molecular weight of which was one-third of that of each parent d-mannan, had only one half of the antitumor activity of the parent d-mannans. This suggests that molecular size is the most important factor for the differences in activity of the polysaccharides of wild and mutant strains.     

Different porphobilinogen-deaminase forms in wild and mutant strains of Saccharomyces cerevisiae. A possible correlation with its segregants behaviour.

1. Different porphobilinogen-deaminase (PBG-D) enzyme forms were found for D 27 and D 27/C6 (HEM R+) strains of Saccharomyces cerevisiae. 2. PBG-D was partially purified and chromatographed on Sephadex G-100 in either the presence or absence of a protease inhibitor. For D 27 only one active peak was observed while for D 27/C6 strain two active peaks were found. 3. A correlation between this differential behaviour and the presence of HEM R+ gene was looked for employing two segregants of one tetrad from D 27 and D 27/C6 mating.     

Pleiotrophic cellular deficiencies conferred by the blm5-1 mutation of Saccharomyces cerevisiae.

Mutational alteration of the BLM5 gene of the model eukaryote, Saccharomyces cerevisiae, confers extreme hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins. Additional properties conferred by the blm5-1 mutation in haploid and diploid strains were investigated for the current report. Only one copy of blm5-1 together with the normal BLM5 allele was sufficient to produce mitotic and meiotic defects in diploids, and greatly increase killing by bleomycin beyond wild type levels. Mitotic growth rates of blm5-1/blm5-1 homozygous mutant strains were slower than wild type or BLM5/blm5-1 heterozygous strains at 30 degrees C, and growth was nearly completely inhibited at 37 degrees C. Meiosis was inhibited at 30 degrees C and 37 degrees C in mutant homozygotes, and at 37 degrees C in BLM5/blm5-1 heterozygotes, while meiosis occurred at equivalent frequencies in wild type strains at both temperatures. Surprisingly, mutant strains were found to associate extremely low quantities of [S-methyl-3H]bleomycin A2, in contrast to normal strains that associated quite high amounts. However, the fractions of the total associated radioactivities that were released from normal and blm5-1 cells were equivalent. These results suggested that the extremely high killing suffered by blm5-1 mutant strains in response to bleomycin treatments results from something other than increased intracellular drug concentrations.     

The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit.

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.     

The phosphorus content of some aquatic macrophytes with special reference to seasonal fluctuations and applications of phosphate fertilizers

Summary Preliminary observations on the increases in phosphorus concentration of seven aquatic plant species, following the addition of superphosphate fertilizer to the water of a Perthshire loch, showed that in only two species, Myriophyllum alterniflorum and Potamogeton praelongus, was the increased uptake significant.Estimations of the phosphorus content of these species in unfertilized water were made over a period of two years and the results demonstrate the existence of a seasonal cycle, related to the development of the plants. Enrichment of Scottish hill lochs with superphosphate produced significant increases in the phosphorus concentration of two species of Myriophyllum, M. alterniflorum and M. spicatum. The increases from fertilizer application in May were not sufficient to offset the seasonal decline which occurs in May and June, but fertilizer application in August produced a significant upward trend.

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Résumé Des observations préliminaires des accroissements de la concentration de phosphore de sept espèces de plantes aquatiques, à la suite de l'addition d'engrais phosphatique à l'eau douce d'un lac en Perthshire, Ecosse, montrèrent qu'en deux espèces seulement l'accroissement à l'absorption de phosphate était significatif.Des estimations du contenu de phosphore de ces espèces à l'eau douce infertilisée ètaient faites au cours d'une période de deux années et les résultats d'émontrent l'existence d'un cycle qui dépend de la saison, connexe au développement des plantes. L'enrichissement de quelques lacs aux montagnes écossaises avec phosphate produisit des accroissements significatifs de la concentration de phosphore de deux espèces de Myriophyllum, Myriophyllum alterniflorum et Myriophyllum spicatum. Les accroissements résultants de l'application d'engrais en mai ne suffisaient pas de compenser le déclin saisonnier qui se présente en mai et juin, mais l'application d'engrais en août produisit une tendance ascendante et significative.

     

Historical view of DNA replication studies, with special reference to Japan

Almost forty years after the key contributions to the field by Okazaki and coworkers that gave rise to the concept of leading and the lagging strand, we are still at the state of uncertainty about the proteins that replicate each strand. Perhaps, one main conclusion that should be drawn from the data currently available is that the protein architecture at the fork is more plastic than originally thought.