Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

Abstract

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes 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. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora

“…In addition to the recently described A-karyotype (Walz & Ku È ck, 1995), we were able to identify a second electrophoretic karyotype (B-karyotype) that differs in size in at least four out of seven chromosomes ( Fig. 1; Table 2).…”

“…Chromosomes were separated in 0.8% agarose (Fast Laneä, FMC, Rockland, ME, USA) gels with 0.5´TBE buffer (pH 8.3) at 12°C using the CHEF Mapperä (BioRad, Richmond, CA, USA). Separation conditions were, unless otherwise speci®ed, identical to those reported by Walz & Ku È ck (1995). Schizosaccharomyces pombe chromosomes (BioRad) were used as a size standard.…”

“…Ku È ck, 1995). In analysing different S. macrospora wildtype and mutant strains from several sources (Table 1), we are able to distinguish two major electrophoretic karyotypes, which we designated A and B (Fig.…”

“…From these measurements, the size of the whole genome of the A-karyotype was estimated to be 39.5 Mb, while we calculated a genome size of 38 Mb for B-karyotype strains. To con®rm that strains presenting the B-karyotype in the PFGE have the same number of chromosomes as the A-karyotype (Walz & Ku È ck, 1995), we compared the results obtained by PFGE with cytological observations. Figure 2 shows a light microscopic photo of the ®rst post-meiotic metaphase from the B-karyotype found in strain K(3346) of S. macrospora.…”

“…In a previous work, Walz & Ku È ck (1995) determined the electrophoretic karyotype of the S. macrospora fertile strain K(18). Using PFGE, they were able to resolve ®ve Keywords: electrophoretic karyotypes; intraspecies polymorphism; pulsed-®eld gel electrophoresis.…”