J. R. Villanueva

Cloning of genes related to exo-β-glucanase production in Saccharomyces cerevislae: characterization of an exo-β-glucanase structural gene

Abstract The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain ( exgl -2) with highly reduced extracellular exo-β-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb Hind III- Xho I fragment. Exg + strains and Exg − mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg + wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg + wt strains. The EXG1 gene transformed Schizosaccharomyces pombe , yielding extracellular EXG activity which showed cross-reactivity with anti- S. cerevisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg + strain. Genetic and Southern analysis of several stable Exg − transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exgl -2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae .

Formation of protoplasts ofFusarium culmorum by strepzyme

Spherical protoplast-like structures can be liberated from hyphae ofFusarium culmorum by the action of an enzyme preparation obtained from the cell-free supernatant of the growth medium ofStreptomyces RA. In a previous publication this lytic system was named “strepzyme”. Treatment was carried out in a citrate phosphate buffer containing 0.8m mannitol or KCl as a stabilizing agent. If the concentration of the stabilizer was lowered to less than 0.2m the protoplasts lysed. These osmotically sensitive structures vary largely in size, are resistant to high speed centrifugation, and stable when kept in the cold. Protoplasts from other molds could also be obtained. The protoplasts could be observed to emerge through pores in the hyphal wall leaving behind delicate but rigid empty cell walls resistant to enzymic digestion and recognizable under the phase-contrast microscope. After sudden and complete lysis of protoplasts some membrane structures were observed. Attempts to obtain electron micrographs of the latter have failed. It was concluded that in the strain ofFusarium studied the strepzyme removes some constituent of the cell wall facilitating the extrusion of the protoplasts.

PARTICIPATION OF DOLICHOL PHOSPHO-MANNOSE IN THE GLYCOSYLATION OF YEAST WALL MANNO-PROTEINS AT THE POLYSOMAL LEVEL

1. Introduction The major yeast wall glycoprotein (yeast mannan) is formed by mannose units linked to polypeptide backbones either through an ,O-glycosydic bond to hydroxyl groups of serine and threonine or by an N- glycosydic bond conecting a residue of asparagine to a di&acetyl-chitobiose unit [ 1,2] . Dolichol-phosphate derivates especially dolichol- phosphomannose (DPM) appear to play an important role [3,6,18] in the transfer of the first mannosyl residue to the hydroxyaminoacids. Guanosine-diphos- phate-mannose (GDP-mannose) acts as the intermediate donor of mannose residues for further elongation of the oligosaccharides [7]. Some of the enzymes involv- ed in the synthesis of specific bonds have also been characterized [6,8,21]. Evidence that the initial glycosylation of glyco- proteins in higher cells occurs at the level of nascent polypeptide has been reported by different groups [9-l 21 and studies carried out in our laboratory have shown that glycosylation of the mannoproteins of

Observations on the Protoplasts of Fusarium culmorum and on their Fusion

SUMMARY: Some characteristics of protoplast extrusion from hyphae of Fusarium culmorum are described. One, two or more protoplasts might be released from one mycelial compartment. The release was stimulated by controlled dilution of the stabilizing solution. NH4Cl and mannitol were the best stabilizers of those tested. Connecting threads between protoplasts were observed; their meaning is discussed. Changes in the growth medium did not affect mycelium sensitivity for protoplast formation. Various stages of a protoplast fusion process were followed. Protoplast fusion usually began by an attraction of two bodies followed by junction and fusion to give a single large body. There was no evidence that these fusions represent a sexual process. Fusion of protoplasts in Fusarium is infrequent.

Heterogeneous glycosylation of the EXG1 gene product accounts for the two extracellular exo-β-glucanases of Saccharomyces cerevisiae

Two exo‐β‐glucanases of glycoprotein nature can be detected in culture supernatants of Saccharomyces cerevisiae cells. These exo‐β‐glucanases show different M r values and kinetic properties, although they are immunologically related. Their carbohydrate content and the electrophoretic mobility of both endoglycosidase H‐treated exo‐β‐glucanases suggest that they share the same protein fraction. Studies at genetic level relate the production of both extracellular exo‐β‐glucanases with the expression of a single‐copy gene in S. cerevisiae. Expression of this gene in another yeast, Schizosaccharomyces pombe, demonstrates that it codes for a protein with exo‐β‐glucanase activity whose heterogeneous N‐glycosylation accounts for both extracellular exo‐β‐glucanases of S. cerevisiae.

Invertase messenger ribonucleic acid in Saccharomyces cerevisiae: Kinetics of formation and decay

Saccharomyces cerevisiae -136ts (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807-814) incubated in the presence of maltose at 23 degrees C (permissive temperature) synthesized the RNA messengers which codify derepressed invertase (an external mannoprotein) and induced alpha-glucosidase (a non-glycosylated internal enzyme). The enzymes were not synthesized if the mutant was transferred to the maltose-containing medium at the moment of incubation at 37 degrees C indicating that the cells had no pools of the specific RNA messengers and that transcription of the DNA was a prerequisite to enzyme synthesis. Cycloheximide inhibited syntheses of the enzymes both at 37 and at 23 degrees C suggesting that the enzymic activities were the result of de novo synthesis of the proteins and did not result from the activation of proenzymes. In derepressed cells the number of invertase mRNA molecules is probably larger than that actually being translated. The half-life of the derepressed invertase mRNA was calculated from the moment that the molecules of RNA messenger were limiting the enzyme synthesis and a value of 30-35 min was estimated. The value found for the basal (repression independent) invertase mRNA was of 45-50 min. The half-life of alpha-glucosidase mRNA was computed following the mathematical procedure described in the Appendix, and a value of 23 min was obtained. These results are consistent with the existence of relatively long-lived RNA messengers involved in the synthesis of extracellular macromolecules.

Yeast Invertase" Subcellular Distribution and Possible Relationship between the Isoenzymes

The intracellular invertase ofSaccharomyces cerevisiae is mainly found in a soluble form (91–95%), while only minor amounts are found bound to the internal (4–8%) and plasma membranes (less than 1%). In the processes of derepression or repression, inhibition of RNA or protein synthesis, or in the presence of 2-deoxy-d-glucose, the levels of the membrane-bound and external activities are modified in a way in which their relation is clear, while the soluble enzyme does not change at all. These results, together with the fact that the membrane-bound and the external enzymes are glycoproteins, suggest a precursor-product relationship between the enzymic forms.

Preparation of protoplast-like structures from conidia ofFusarium culmorum

Protoplast-like structures have been formed by digestion of the cell walls ofFusarium culmorum conidia by lytic enzyme preparations ofMicromonospora AS. Under the test conditions extrusion of the protoplasts was not observed. It seems that digestion of the cell wall occurs in different stages. Digestion of the septa preceded the formation of protoplasts of the individual cells of the multicellularF. culmorum conidia. A few protoplasts survived the lytic enzyme treatment. “Protoplasts” obtained from conidia are much more stable than those obtained from young hyphae and were able to germinate with the formation of normal mycelium. Lysis of some of the protoplast bodies led to the formation of a membranous structure. The protoplasts derived from each of the constituent cells of the conidia could be isolated with the micromanipulator. No differences were found in the ability of the isolated cells to germinate.

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.

Budding in Saccharomyces cerevisiae: Formation of the cross-wall

Formation of the cross-wall during budding of Saccharomyces cerevisiae Hansen was investigated by electron microscopy of cells treated with toluene-ethanol solutions. This treatment halts cellular metabolism and therefore labile membranous structures can be visualized. Appearance of these structures and their modifications during different stages of budding suggest that they might be involved in synthesis and secretion of wall material. The possible relationship between these membranous structures and dictyosomes is discussed.