Some factors affecting transformation of Aspergillus nidulans-Problems and progress

Some factors affecting transformation of Aspergillus nidulans Problems and progress. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol34/iss1/16 pen-1: perithecial neck-1 VII. Linked csp-2 (4%) Perithecia are barren when pen-1 is used as female, even if the fertilizing parent is pen^+. An ostiole is present, but no beak. No ascospores are extruded. A few croziers and asci are present within the beakless perithecia, and a few mature ascospores may be formed. Perithecia are fully fertile and morphologically normal in the reciprocal cross, where pen-1 is used as male to fertilize pen^+. Development is about equally abnormal in pen female x pen^+ male and in pen x pen. Perithecia are normal and fertile when the female parent is heterokaryotic for pen in the cross (pen-1;un-3 ad-3 nit-2 A + pen^+; a^m1 ad-3B cyh-1) female x pen-1 a male, even though all asci are homozygous for the mutant pen-1 allele. Allele DL413 was obtained and described by DeLange and Griffiths 1980 Genetics 96:367-378. Cytology by N.B. Raju. Mapping by Perkins, Pollard and F.M. Delagi ufa(P73B118): unsaturated fatty acids IVR. Linked to met-5 (7%). (Likely a recurrence of ufa-1/ufa-2, which were mapped to IV or V by Scott 1977, J. Bacteriol. 130:1144-1148) Requirement and stability similar to those described for ufa-1 and ufa-2, stocks of which have been lost. Responds strongly to oleic acid and Tween 80 (oleic + palmitic), not to palmitic, Tween 40 (palmitic), stearic or linolenic acid, and weakly to linoleic (S. Brody, personal communication). Grows well on 1% Tween 80 supplement, but vegetatively unstable. Reversions produce conidiating sectors. Maintained reliably on minimal medium in heterokaryon with a^m1 ad-3B cyh-1. Unripe, light colored ascospores are probably enriched for revertants. Reliable mapping should be based on ufa^progeny. Perithecia are barren in ufa x ufa crosses (made using heterokaryons). Dept. of Biological Sciences, Stanford University, Stanford, CA 94305 Shawcross, S.G., P. Hooley The high frequency transformation vector pDJB3 (Ballance and Turner 1985 Gene 36:321-331) and P. Strike has already proved successful in the cloning of genes affecting both carbon metabolism and spore Some factors affecting transformadevelopment (Ballance and Turner M.G.G. 202:271275). We are currently attempting to clone genes tion of Aspergillus nidulans affecting DNA repair in Aspergillus nidulans using this plasmid to generate genomic DNA banks. Problems and progress Initial problems encountered in poor consistency of transformation have allowed us to identify some of the variables in this system. Crucial to the transformation of A. nidulans is the production of viable protoplasts for introduction to plasmid vectors in the presence of polyethylene glycol (PEG). Using the standard technique of Ballance and Turner (1985) extensive vacuolation of protoplasts was often observed and regeneration frequencies were low (frequency <1%). Increasing the molarity of buffering KCl from 0.6 M to 0.9 M in protoplasting and regeneration media did not, however, markedly improve protoplast regeneration. Protoplasts released by enzymatic digestion of hyphal "mats" vary considerably in size. Size fractionation using "Milipore" filters indicated that protoplasts in the size range 5-8 um (diameter) showed higher regeneration frequencies than those smaller or larger. Table I illustrates some of the techniques used to improve protoplast regeneration. Clearly, while consistent improvements in protoplast regeneration can be achieved, individual experiments still show great variation. Protoplast suspensions of the standard A. nidulans strain G191 prepared on different occasions using the standard procedure showed variable efficiencies of transformationthe maximum achieved being 9000 per ug DNA (13.4% viable protoplasts transformed) down to a negligible frequency, with around 400 per ug DNA (0.29% viable protoplasts transformed) being a more typical value. Individual plasmid preparations prepared in an E. coli (rec^-) host (HBlOl) differed markedly in transformation efficiency of G191 even though each preparation was "clean" enough to be linearized with EcoRI. An additional phenol extraction of proteins followed by an extra CsCl gradient step may be advisable to rigorously purify plasmid DNA. Table 1: A Summary of Protoplast Regeneration Techniques Treatment % Regeneration (range, 3 or 4 replicates) Medium, buffered with 0.6 M KCl + 50 mM CaCl2 (standard method) 0.5 1.0 Use of cultures grown from regenerated protoplasts Pre-incubation in 100 ug/ml 2 deoxy-D-glucose after growth on complete medium Growth of mycelial mat on medium containing 100 ug/ml 2 deoxy-D-glucose* 5.0 8.0 39.0 64.0 41.0 48.0 Growth of myceliel mat on medium containing 0.4 M NH4Cl 60.0 77.0 * Foury and Goffeau 1973 J. Gen. Micro. 75:227-229 Figure 1 demonstrates that PEG 6000 is more effective than PEG 4000 (both supplied by BDH Chemicals Ltd. Poole, England) at transforming each of two strains, G191 and M, a recombinant strain containing pyrG (for selection of pyrimidine prototrophic transformants) and two mutations increasing sensitivity to alkylating agents. There is no significant difference in the transforming ability of these two strains. Effective screening and sampling procedures for complementation tests with cloned genes should take into account the asynchronous development of transformed colonies. Further experiments revealed that transformed protoplasts could regenerate efficiently on the surfaces of agar plates (containing 0.6 M KCl) although regeneration occurred at higher frequencies in agar overlays. Prolonged treatments in PEG 6000 (up to an extra 20 minutes) appeared not to alter transformation efficiency.

Shawcross, S.G., P. Hooley The high frequency transformation vector pDJB3 (Ballance and Turner 1985 Gene 36:321-331) and P. Strike has already proved successful in the cloning of genes affecting both carbon metabolism and spore Some factors affecting transforma-development (Ballance and Turner M.G. G. 202:271-275). We are currently attempting to clone genes tion of Aspergillus nidulans -affecting DNA repair in Aspergillus nidulans using this plasmid to generate genomic DNA banks. Problems and progress Initial problems encountered in poor consistency of transformation have allowed us to identify some of the variables in this system.
Crucial to the transformation of A. nidulans is the production of viable protoplasts for introduction to plasmid vectors in the presence of polyethylene glycol (PEG). Using the standard technique of Ballance and Turner (1985) extensive vacuolation of protoplasts was often observed and regeneration frequencies were low (frequency <1%). Increasing the molarity of buffering KCl from 0.6 M to 0.9 M in protoplasting and regeneration media did not, however, markedly improve protoplast regeneration. Protoplasts released by enzymatic digestion of hyphal "mats" vary considerably in size. Size fractionation using "Milipore" filters indicated that protoplasts in the size range 5-8 um (diameter) showed higher regeneration frequencies than those smaller or larger. Table I illustrates some of the techniques used to improve protoplast regeneration. Clearly, while consistent improvements in protoplast regeneration can be achieved, individual experiments still show great variation.
Protoplast suspensions of the standard A. nidulans strain G191 prepared on different occasions using the standard procedure showed variable efficiencies of transformationthe maximum achieved being 9000 per ug DNA (13.4% viable protoplasts transformed) down to a negligible frequency, with around 400 per ug DNA (0.29% viable protoplasts transformed) being a more typical value.
Individual plasmid preparations prepared in an E. coli (rec^-) host (HBlOl) differed markedly in transformation efficiency of G191 even though each preparation was "clean" enough to be linearized with EcoRI. An additional phenol extraction of proteins followed by an extra CsCl gradient step may be advisable to rigorously purify plasmid DNA.

Use of cultures grown from regenerated protoplasts
Pre-incubation in 100 ug/ml 2 deoxy-D-glucose after growth on complete medium Growth of mycelial mat on medium containing 100 ug/ml 2 deoxy-D-glucose* demonstrates that PEG 6000 is more effective than PEG 4000 (both supplied by BDH Chemicals Ltd. Poole, England) at transforming each of two strains, G191 and M, a recombinant strain containing pyrG (for selection of pyrimidine prototrophic transformants) and two mutations increasing sensitivity to alkylating agents. There is no significant difference in the transforming ability of these two strains. Effective screening and sampling procedures for complementation tests with cloned genes should take into account the asynchronous development of transformed colonies.
Further experiments revealed that transformed protoplasts could regenerate efficiently on the surfaces of agar plates (containing 0.6 M KCl) although regeneration occurred at higher frequencies in agar overlays. Prolonged treatments in PEG 6000 (up to an extra 20 minutes) appeared not to alter transformation efficiency. When problems are experienced in achieving transformation of A. nidulans, it seems likely that further variables will be identified. For example, different batches of PEG manipulation of Streptomyces -a laboratory manual, John Innes Foundation