| Author/Editor | Rozman, Damjana | |
| Title | Heterologna transformacija in steroidni metabolizem filamentozne glive Cochliobolus lunatus | |
| Type | monografija | |
| Place | Ljubljana | |
| Publisher | Univerza v Ljubljani, Medicinska fakulteta | |
| Publication year | 1993 | |
| Volume | str. 97 | |
| Language | slo | |
| Abstract | Since in former transformation experiments with the filamentous fungus Coćhliobolus lunatus m118 morphologically and metabolically altered colonies were obtained, which were not easily genetically characterized (Dermastia et al., FEMS Microbiol. Lett. 77:145-150, 1991; Rozman and Komel, Curr. Genet. 22:123-127, 1992) we decided to define the genome structure as well as the ultrastructure of C. lunafus in comparison with the single and genetically better characterized species of the anamorphic strain Curvularia lunata. The electron microscopic studies revealed the ultrastructure of both strains to be significantly different. While C. lunatus m118 grows in hyphal form characterized by a thick cell wall and numerous lipid bodies, C. lunata AT46 forms thinner hyphae of various sizes and large oval spores. Using pulse field gel electrophoresis, the genome of C. lunatus m118 was resolved in 12 chromosomes, with the largest one of 5.2 Mb and the smallest one of 1.5 Mb. The size of the genome was estimated to be 35.1 Mb q 10 per cent . Electrophoretic karyotypes of C. lunatus m118 (this work) and C. lunata AT46 (Osiewacz, H., Ridder R., Curr. Genet. 20:151-155,1991) as well as RAPD-PCR analysis with primers (GTG)5 , (GATA)6 and with combination of both primers, indicate a close genetic relationship of both morphologically distinct microorganisms. Filamentous fungus Cochliobolus lunatus m118 was successfully transformed by electroporation with the heterologous plasmid pUT 737, harboring Sh ble gene as a dominant selection marker under the control of strong TR1 promoter from Trichod derma reesei (Calmels et al., J. Biotechnol. 17:51-66, 1991). The best transformation results were obtained when the time constant z reached the value of 3.5 s - 4.5 s, which was attained using an electric field of 1000 V/cm or 2250 V/cm, a capacity of 25 micro F, and a resistanee of 1000 ohms. A new method for the isolation of genomic DNA from filamentous fungi was developed.(trunc.) | |
| Descriptors | ASCOMYCETES TRANSFORMATION, GENETIC GENOME, FUNGAL DNA, FUNGAL MICROSCOPY, ELECTRON KARYOTYPING GENETIC VECTORS GENETIC MARKERS HYBRIDIZATION PLASMIDS BLEOMYCINS |