Introduction of hygromycin B resistance into Schizophyllum commune: preferential methylation of donor DNA.
Cotransformation of a trp1 strain of Schizophyllum commune with the homologous TRP gene and the Escherichia coli HPT gene was used to study the feasibility of transformation of S. commune to hygromycin B resistance. Southern blot analysis showed that 75% of the TRP transformants contained multiple integrated copies of the HPT gene. However only 7% of the transformants were resistant to 25 micrograms/ml hygromycin B and direct selection for hygromycin B resistance was hampered by the high incidence of spontaneously arising resistant colonies. Rescue of the HPT gene was possible with E. coli JA221 (mcr-) but not with JM83, suggesting methylation of the integrated donor DNA. Isoschizomer analyses confirmed heavy methylation in the HPT gene and flanking vector sequences but not in the homologous donor TRP gene and its flanking vector sequences. Also cotransforming S. commune Sc4 gene and flanking vector sequences were not methylated. A fusion between the S. commune TRP1 and the E. coli HPT genes resulted in only slight or no methylation of both vector and HPT sequences and in a higher hygromycin B resistance level. This suggests that transformation with DNA exclusively containing foreign sequences results in integration into regions where methylation occurs, possibly entailing poor transcription. Methylation of the HPT gene was also indicated by the stimulation of growth by 5-azacytidine of transformants on hygromycin B containing medium.
Department of Plant Biology, University of Groningen, Haren, The Netherlands.
H
H
Mooibroek
A G
AG
Kuipers
J H
JH
Sietsma
P J
PJ
Punt
J G
JG
Wessels
D001374:Azacitidine / Q000378:metabolism; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006921:Hygromycin B / Q000494:pharmacology*; D008745:Methylation; D012567:Schizophyllum / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D006921:Hygromycin B; D003596:Cytosine; D001374:Azacitidine