Akileswaran L, Alic M, Clark E K, Hornick J L, Gold M H
Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton 97006-1999.
Curr Genet. 1993;23(4):351-6. doi: 10.1007/BF00310898.
Uracil auxotrophs of Phanerochaete chrysosporium were isolated using 5-fluoroorotate resistance as a selection scheme. The ura3 auxotrophs deficient in orotidylate decarboxylase and ura5 auxotrophs deficient in orotate phosphoribosyl transferase were characterized by enzyme assays and complementation tests. The ura5 auxotrophs were transformed to prototrophy with the ura5 gene from the ascomycete Podospora anserina. The ura3 auxotrophs were transformed to prototrophy with the ura3 gene from the basidiomycete Schizophyllum commune. The P. chrysosporium ura3 gene was isolated from a lambda EMBL3 genomic library using the S. commune ura3 gene as a probe. A 6.6-kb fragment incorporating the ura3 gene was subcloned into Bluescript SK+(pURA3.1) and used to transform P. chrysosporium ura3 auxotrophic strains. The pURA3.1 insert was mapped for restriction sites and the approximate location of the ura3 gene within the insert was determined. Double auxotrophic strains were transformed with either of two marker genes and the resulting single auxotrophic strains were crossed to demonstrate genetic recombination between two nuclei of identical genetic background.
利用对5-氟乳清酸的抗性作为选择方案，分离出了黄孢原毛平革菌的尿嘧啶营养缺陷型菌株。通过酶活性测定和互补试验对缺乏乳清酸核苷脱羧酶的ura3营养缺陷型菌株和缺乏乳清酸磷酸核糖转移酶的ura5营养缺陷型菌株进行了表征。ura5营养缺陷型菌株用来自子囊菌粗糙脉孢菌的ura5基因转化为原养型。ura3营养缺陷型菌株用来自担子菌裂褶菌的ura3基因转化为原养型。以裂褶菌的ura3基因为探针，从λ EMBL3基因组文库中分离出黄孢原毛平革菌的ura3基因。将包含ura3基因的一个6.6kb片段亚克隆到pBluescript SK+(pURA3.1)中，并用于转化黄孢原毛平革菌的ura3营养缺陷型菌株。对pURA3.1插入片段进行了限制性酶切位点作图，并确定了ura3基因在插入片段中的大致位置。用两个标记基因中的任何一个转化双营养缺陷型菌株，然后将得到的单营养缺陷型菌株进行杂交，以证明相同遗传背景的两个细胞核之间的基因重组。
