TRANSFORMATION OF SORDARIA-MACROSPORA TO HYGROMYCIN-B RESISTANCE - CHARACTERIZATION OF TRANSFORMANTS BY ELECTROPHORETIC KARYOTYPING AND TETRAD ANALYSIS

The ascomycete Sordaria macrospora was transformed using different pla smid molecules containing the bacterial hygromycin B resistance gene ( hph) under the control of different expression signals. The highest tr ansformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream regio n of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector co pies are integrated as tandem repeats into the S. macrospora chromosom es and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance ( hygR) is not correlated with the number of integrated vector molecules . Electrophoretic karyotyping was used to further characterize S. macr ospora transformants. Five chromosomal bands were separated by pulsed- field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transf ormants, major rearrangements were detected. Transformants were sexual ly propagated to analyze the fate of the heterologous vector DNA. Alth ough the hygR phenotype is stably maintained during mitosis, about a t hird of all lines tested showed loss of the resistance marker gene aft er meiosis. However, as was concluded from electrophoretic karyotyping , the resistant spores showed a Mendelian segregation of the integrate d vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformatio n tagging, or the molecular mapping of chromosomal loci in S. macrospo ra