Froeliger E H, Muñoz-Rivas A M, Specht C A, Ullrich R C, Novotny C P
Department of Microbiology, University of Vermont, Burlington 05405.
Curr Genet. 1987;12(7):547-54. doi: 10.1007/BF00419565.
We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ura1, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trp1, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, ade5, pab1, trp1, and ura1 mutations were recovered.
我们已经开发出一种常规方法，可直接从担子菌真菌裂褶菌中分离基因。通过转化互补裂褶菌突变，利用基因组基因文库中的质粒DNA分离出了五个特定基因。这些突变菌株在合成腺嘌呤（ade2和ade5）、尿嘧啶（ura1，编码乳清酸核苷-5'-磷酸脱羧酶；OMP脱羧酶）、色氨酸（trp1，编码吲哚-3-甘油磷酸合成酶；IGPS）或对氨基苯甲酸（pab1）方面存在缺陷。在每种情况下，Southern分析表明，向原养型的转化伴随着载体序列整合到裂褶菌突变体的基因组中。来自转化体的总DNA经酶切、重新连接后，用于转化大肠杆菌。从大肠杆菌中回收氨苄青霉素抗性质粒，并测试其转化裂褶菌相应突变体的能力。回收了互补ade2、ade5、pab1、trp1和ura1突变的质粒。
