Abstract
Uracil auxotrophs of Phanerochaete chrysosporium were isolated using 5-fluoroorotate resistance as a selection scheme. The ura3 auxotrophs deficient in orotidylate decarboxylase and ura5 auxotrophs deficient in orotate phosphoribosyl transferase were characterized by enzyme assays and complementation tests. The ura5 auxotrophs were transformed to prototrophy with the ura5 gene from the ascomycete Podospora anserina. The ura3 auxotrophs were transformed to prototrophy with the ura3 gene from the basidiomycete Schizophyllum commune. The P. chrysosporium ura3 gene was isolated from a γEMBL3 genomic library using the S. commune ura3 gene as a probe. A 6.6-kb fragment incorporating the ura3 gene was subcloned into Bluescript SK+(pURA3.1) and used to transform P. chrysosporium ura3 auxotrophic strains. The pURA3.1 insert was mapped for restriction sites and the approximate location of the ura3 gene within the insert was determined. Double auxotrophic strains were transformed with either of two marker genes and the resulting single auxotrophic strains were crossed to demonstrate genetic recombination between two nuclei of identical genetic background.
Similar content being viewed by others
References
Alani E, Liang C, Kleckner N (1987) Genetics 116:541–545
Alic M, Gold MH (1985) Appl Environ Microbiol 50:27–30
Alic M, Letzring C, Gold MH (1987) Appl Environ Microbiol 53:1464–1469
Alic M, Kornegay JR, Pribnow D, Gold MH (1989) Appl Environ Microbiol 55:406–411
Alic M, Clark EK, Kornegay JR, Gold MH (1990) Curr Genet 19:305–311
Alic M, Mayfield MB, Akileswaran L, Gold MH (1991) Curr Genet 19:491–494
Alic M, Gold MH (1991) In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, San Diego, pp 319–341
Asch DK, Kinsey JA (1990) Mol Gen Genet 221:37–43
Bégueret J, Razanamparany V, Perrot M, Barreau C (1984) Gene 32:487–492
Binninger DM, Le Chevanton L, Skrzynia C, Shubkin CD, Pukkila PJ (1991) Mol Gen Genet 227:245–251
Boeke JD, LaCroute F, Fink GR (1984) Mol Gen Genet 197:345–346
Boeke JD, Truehart J, Natsoulis G, Fink GR (1987) Methods Enzymol 154:164–175
Brown JA, Alic M, Gold MH (1991) J Bacteriol 173:4101–4106
Bumpus JA, Aust SD (1987) BioEssays 6:166–170
Buswell JA, Odier E (1987) CRC Critical Rev Biotechnol 6:1–60
Esser K (1966) Incompatibility. In: Ainsworth GC, Sussman AS (eds) The Fungi: an advanced treatise, vol II. Academic Press, New York, pp 661–676
Fincham JRS (1989) Microbiol Rev 53:148–170
Froeliger EH, Ullrich RC, Novotny CP (1989) Gene 83:387–393
Godfrey BJ, Mayfield MB, Brown JA, Gold MH (1990) Gene 93:119–124
Gold MH, Cheng TM, Mayfield MB (1982) Appl Environ Microbiol 44:996–1000
Gold MH, Wariishi H, Valli K (1989) In: Whitaker JR, Sonnet PE (eds) Biocatalysis in agricultural biotechnology. ACS, Washington DC, pp 127–140
Grimm C, Kohli J, Murray J, Maundrell K (1988) Mol Gen Genet 215:81–86
Hammel KE (1989) Enzyme Microb Technol 11:776–777
Hynes MJ (1989) Exp Mycol 13:196–198
Kelly R, Miller SM, Kurtz MB, Kirsch DR (1987) Mol Cell Biol 7:199–207
Kirk TK, Farrell RL (1987) Annu Rev Microbiol 41:465–505
Krejčí R (1987) Folia Microbiol 32:177–180
Krejčí R, Homolka L (1991) Appl Environ Microbiol 57:151–156
Kronstad JW, Wang J, Covert SF, Holden DW, McKnight GL, Leong SA (1989) Gene 79:97–106
Liwicki R, Paterson A, MacDonald MJ, Broda P (1985) J Bacteriol 162:641–644
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Mellon FM, Casselton LA (1988) Curr Genet 14:451–456
Miller BL, Miller KY, Roberti KA, Timberlake WE (1987) Mol Cell Biol 7:427–434
de Montigny J, Kern L, Hubert JC, Lacroute F (1990) Curr Genet 17:105–111
Prabhakararao K, Jones ME (1975) Anal Biochem 69:451–467
Raeder U, Broda P (1986) EMBO J 51:1125–1127
Razanamparany V, Bégueret J (1986) Curr Genet 10:811–817
Razanamparany V, Bégueret J (1988) Gene 74:399–409
Smith MH, Gold MH (1979) Appl Environ Microbiol 37:938–942
Thompson W, Broda P (1987) Trans Br Mycol Soc 89:285–294
Turcq B, Bégueret J (1987) Gene 53:201–209
Valli K, Gold MH (1991) J Bacteriol 173:345–352
Valli K, Warriishi H, Gold MH (1992) J Bacteriol 174:2131–2137
Wariishi H, Valli K, Gold MH (1989) Biochemistry 28:6017–6023
Wariishi H, Valli K, Gold MH (1991) Biochem Biophys Res Commun 176:269–275
Yoshimoto A, Amaya T, Kobayashi K, Tomita K (1978) Methods Enzymol 51:69–74
Author information
Authors and Affiliations
Additional information
Communicated by K. Esser
Rights and permissions
About this article
Cite this article
Akileswaran, L., Alic, M., Clark, E.K. et al. Isolation and transformation of uracil auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium . Curr Genet 23, 351–356 (1993). https://doi.org/10.1007/BF00310898
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00310898