Biosynthesis of the yeast cell wall: Selective assays and regulation of some mannosyl transferase activities

Assays have been developed for some transfer reactions involved in the synthesis of Saccharomyces cerevisiae wall mannoproteins, both in a particulate preparation in the presence of EDTA or Triton X-100, and after lipid extraction with chloroform-methanol at -20C.The mannosyl transferase activities were also studied in cells made permeable to GDP-mannose by toluene-ethanol treatment (in situ). In these permeabilized cells, the glycosylating reactions dependent on lipid carriers (dolichol derivatives) did not function, but those independent of them were unaffected.The lipid-independent mannosyl transferase activities were partially inhibited by nucleotide diphosphates probably in a competitive manner. Increase of the nucleotide diphosphate pool in vivo might slow down the speed of the transfer reactions carried out by the mannan synthetase system.     

Human platelet arginase

Summary We report here, for the first time the presence of arginase in human platelets. This enzyme has been partially purified and some of its properties studied. Its biological significance and its involvement in polyamine biosynthesis are considered.     

Evolution of the nucleotide sequence of influenza virus RNA segment 7 during drift of the H3N2 subtype

The complete genetic information contained in the influenza virus RNA segment 7 of the A/Bangkok/ $\text{1}\text{79}$ (H3N2) strain has been cloned by in vitro synthesis of the complementary dsDNA and its insertion into plasmid pBR322. The nucleotide sequence of the viral RNA segment has been determined from the cDNA insert. It is 1027 nucleotides long, and contains two open reading frames, as shown for other influenza virus strains. When compared with the previously published sequence for the A/Udorn/72 (H3N2) strain, 15 nucleotide exchanges are observed, most of them silent mutations, and only two causing amino acid changes in each of the M1 and M2 protein sequences.     

Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology

The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37°C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours.If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37°C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident.These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.This work was supported in part by a grant from la Comisión de Investigación Científica y Técnica of the Spanish Ministerio de Educación y Ciencia (Project no. 4593-1980).     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Decrease of phosphofructokinase activity in relation to the pathogenesis of triorthocresyl-phosphate-induced delayed neuropathy.

The in vivo effect of a single dose of the neuropathic compound triorthocresyl-phosphate (TOCP) on phosphofructokinase (PFC, E.C. 2.7.1.11) and its relation with the initiation step (inhibition and aging of neuropathy target esterase, NTE) in the TOCP-induced delayed neuropathy have been studied. Hens were treated with a neurotoxic dose of TOCP (500 mg/kg, p.o.) and with a protective compound (Phenylmethanesulfonyl fluoride, PMSF, 30 mg/kg s.c.) in different combinations: TOCP, TOCP + PMSF, PMSF + TOCP and PMSF. PFK activity was determined in brain and sciatic nerve 1, 3, 7 and 15 days after treatment. PFK activity decreased in sciatic nerve 15 days after dosing with TOCP or TOCP + PMSF. When animals were dosed with the protective agent (PMSF) alone or before administering the neurotoxic compound, PFK activity was unaltered and clinical signs of neuropathy were absent. The data presented here suggest that phosphofructokinase is involved in the pathogenesis of the neuropathy induced by TOCP.     

Asymmetrical correlated responses to selection under an infinitesimal genetic model

Summary Asymmetry in correlated responses to selection is expected when more than one cycle of selection is practised due to changes in genetic parameters produced by selection. In large populations, under the infinitesimal model these changes are due to linkage disequilibrium generated by selection and not to gene frequency changes. This study examines the conditions under which asymmetrical correlated responses are to be expected when an infinitesimal model is considered. Asymmetrical correlated responses in two traits in respect to which trait is selected are expected if the two traits have different heritabilities. Predicted asymmetry increases with the absolute value of the genetic correlation between the two traits, the difference between the two heritabilities, the intensity of selection and the number of generations of selection. Linkage disequilibrium generated by selection should be taken into account in explaining asymmetrical correlated responses observed in selection experiments.     

Adsorption of bacteriophage phi 29 to Bacillus subtilis through the neck appendages of the viral particle.

Phage phi 29 particles produced under restrictive conditions by mutants in gene 12 have normal amounts of all of the structural proteins except the appendage protein, p12*, which is missing. These particles are not infective and do not adsorb to Bacillus subtilis cells. By in vitro complementation of 12- particles with extracts containing protein p12* or with purified protein p12*, the defective particles could bind the appendage protein and become infective and able to adsorb to bacteria. Therefore, the neck appendages of phage phi 29, formed by protein p12*, are involved in the interaction of the phage with the cell wall receptors. Protein p12*, purified in its native state, competed with wild-type phage for adsorption to bacteria. Also, protein p12* could displace adsorbed phage from bacteria. Since the displaced phage was infective, protein p12* does not seem to be modified after phage adsorption.     

Regulation by aromatic amino acids of the biosynthesis of candicidin by Streptomyces griseus

The biosynthesis by Streptomyces griseus of candicidin, an aromatic polyene macrolide antibiotic, was inhibited by L-tryptophan, L-phenylalanine and, to a lesser degree, by L-tyrosine. A mixture of the three aromatic amino acids inhibited candicidin biosynthesis to a greater extent than did each amino acid separately. L-Tryptophan strongly inhibited the incorporation of the labelled precursors propionate or 4-aminobenzoic acid into candicidin. Incorporation of propionate into candicidin was 50% inhibited by 2.5 mM-tryptophan. Inhibition by tryptophan did not require protein synthesis as the same effect was observed in cells in which protein synthesis was prevented by chloramphenicol. The inhibitory effect of L-tryptophan was partially reversed by exogenous 4-aminobenzoic acid suggesting that this effect is exerted at the level of 4-aminobenzoic acid synthase.     

Inositol deficiency in Saccharomyces cerevisiae NCYC 86

When Saccharomyces cerevisiae NCYC 86, an inositol dependent strain, is grown at suboptimal concentrations of inositol, the buds are apparently unable to separate from the parent cells. Thin sections of such cells show an irregularly thickened cell wall. These morphological features may be due to a continuation or increase in the production of glucan while the synthesis of DNA, RNA, phospholipids and protein is greatly inhibited.