Cathy S. Taft

Inhibition of Neurospora crassa growth by a glucan synthase-1 antisense construct

Abstract. We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)β-glucan synthase [E.C. 2.4.1.34; UDP glucose: 1,3-β-D-glucan 3-β-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)β-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2 [qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction in growth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)β-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)β-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.

Optimal conditions for the release of protoplasts ofNeurospora using Novozym 234

Abstract The optimal conditions for the release of protoplasts from 18-h-hyphae of Neurospora crassa using Novozym 234 were 120-minute incubations at 25°C and 10 mg/ml Novozym. Protoplasts were released from all parts of hyphae and were found not to contain surface-bound cell wall material. About 10% of Novozym-derived protoplasts were viable.

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

Abstract 1,3-β- d -Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aKm,app of 0.75 ± 0.05 mM, and a Vmax,app of 35 ± 1 enzyme units/mg protein.

(1–3)-β-Glucan synthesis ofNeurospora crassa

Abstract(1–3)-β-d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.

In Vitro Inhibition of Stable 1,3-β-D-Glucan Synthase Activity from Neurospora

AbstractGlucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1 % 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, pH 7.4, containing 5mM EDTA, I mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 μM GTP, 1 mM DTT, lOmM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4°C, and a Km app of 1.66 + 0.28mM.Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-β-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki app of each inhibitor determined. It seems likely that this stable pr...