Vloga proteinske kinaze C pri glivi Aspergillus niger : magistrska naloga
Labanac, Nataša
Due to the lack of information about PKC in filamentous fungi, we decided to isolate mutants with different numbers of pkcA genes with molecular biology techniques. To isolate the transformants with ...multiple copy numbers of pkcA genes we contransformed the protoplast with two plasmids, one carrying a selective marker argB and the other pkcA gene. We analysed the transformants, which grew on selective media without arginin for the number of pkcA genes with Southern blotting. We isolated a transformant with two copies (M1) and one with several copies of pkcA gene (M3). We also demonstrated the differencebetween the parental strain and the M1 and M3 transformants by measuring enzyme activity and also by analysing the level of expression of pkcA gene in differenttransformants using RT-PCR. In order to determine which external stimuli activate PKC, we exposed transformants to different environmental stresses and observed the changes in morphology of mycelia afterwards. We also tried to isolate a mutant of A. niger with silenced gene coding for PKC. We synthesised plasmids pAR15 and pPY35, with partially deleted non-functional pkcA gene. The pkcA gene was disrupted by replacing theinternal coding region with the fragment, containing argB+ marker (pAR15) or pyrA+ marker (pPY35). We transformed A. niger protoplasts with each plasmid. An exogenous DNA has to integrate into fungal chromosome for its maintenance. In case of homologous interaction between the native pkcA gene inthe chromosome and the disrupted analogue from plasmid, resulting in gene conversion, we can expect a knock-out mutation of pkcA gene. (Abstract truncated at 2000 characters).
Type of material - master's thesis
Publication and manufacture - Ljubljana : [N. Labanac], 2001
National Institute of Biology and BF, Department of Biology, Ljubljana
BF oddelek za biologijo mg 577.2 LABANAC N. Vloga IN: 0025465
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