Walz M, Kück U
Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Curr Genet. 1995 Dec;29(1):88-95. doi: 10.1007/BF00313198.
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.
使用不同的质粒分子对子囊菌大孢粪壳菌进行转化，这些质粒分子含有在不同表达信号控制下的细菌潮霉素B抗性基因（hph）。使用载体pMW1获得了最高的转化频率。在这个质粒分子上，hph基因的表达由来自半知菌产黄顶头孢霉的异青霉素N合成酶基因（pcbC）的上游区域指导。Southern分析表明，载体拷贝以串联重复的形式整合到了大孢粪壳菌的染色体中，并且重复序列在减数分裂过程中很可能不会因甲基化而失活。此外，潮霉素B抗性（hygR）与整合的载体分子数量无关。使用脉冲场凝胶电泳（PFGE）对大孢粪壳菌转化体进行电泳核型分析，以进一步表征其特征。通过PFGE分离出五条染色体带，代表七条染色体，基因组总大小为39.5Mb。杂交分析揭示了载体DNA异位整合到不同的染色体中。在少数转化体中，检测到了主要的重排。对转化体进行有性繁殖，以分析异源载体DNA的命运。尽管hygR表型在有丝分裂过程中稳定维持，但在所有测试的菌株中，约有三分之一在减数分裂后显示出抗性标记基因的丢失。然而，从电泳核型分析得出的结论是，抗性孢子在至少连续三代中显示出整合的载体分子的孟德尔分离。我们的数据表明，异源标记基因可用于大孢粪壳菌的转化标签或染色体位点的分子定位。
