A homologous gene-reporter system for the basidiomycete Schizophyllum commune based on internally deleted homologous genes.
Problems encountered in our attempts to achieve expression of heterologous genes, driven by ascomycetous regulatory sequences, in homobasidiomycetes led us to develop a gene-reporter system based on the expression of homologous genes in Schizophyllum commune. Internal deletions were made in the coding sequences of the regulated Sc4 gene and the constitutively expressed GPD gene. After introduction of these constructs into S. commune it was found that the expected truncated transcripts were produced. The internally deleted Sc4 gene, containing 1140bp of upstream and 200 bp of downstream sequences, was only expressed in dikaryons at the time of fruiting (as was the resident Sc4 gene) but not at all in monokaryons, indicating that the construct contained all regulatory sequences necessary and sufficient to confer control by the mating-type genes and expression during fruiting. The internally deleted GPD gene, containing 1300 bp of upstream and 150 bp of downstream sequences, was expressed both in monokaryons and dikaryons at levels similar to those of the resident GPD gene, indicating that all sequences necessary for proper expression were present. This reporter-gene system may be applicable to the analysis of cis-regulatory sequences of these genes. Furthermore, heterologous genes may be inserted into the well-expressed GPD deletion construct to obtain expression of such genes in S. commune and possibly in other homobasidiomycetes.
Schuren
F H
FH
Department of Plant Biology, University of Groningen, Haren, The Netherlands.
Harmsen
M C
MC
Wessels
J G
JG
D015152:Blotting, Northern; D015139:Blotting, Southern; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D005809:Genes, Regulator; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012567:Schizophyllum / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D012331:RNA, Fungal