Manavathu E K, Suryanarayana K, Hasnain S E, Leung W C
Department of Medical Microbiology & Infectious Diseases, University of Alberta, Edmonton, Canada.
J Gen Microbiol. 1988 Jul;134(7):2019-28. doi: 10.1099/00221287-134-7-2019.
A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2 microns. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the transformant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.
利用嵌合质粒载体pSV2neo2微米，为水生丝状真菌两性绵霉开发了一种DNA介导的转化系统。在软琼脂上，从转化的原生质体中再生出对新霉素类似物硫酸G-418有抗性的菌丝菌落。对转化细胞DNA的Southern印迹分析产生了多条杂交带，表明载体DNA在多个位点整合到宿主基因组中。Northern印迹分析显示转化体中存在三种APHII基因特异性转录本，表明对G-418的抗性表型是由于APHII基因的表达。存在意外大尺寸的多个RNA转录本，表明RNA起始和/或终止受SV40启动子以外的调控元件控制。用来自真菌转化体的总DNA制剂转化大肠杆菌细胞回收的质粒DNA显示出相当大的DNA重排。然而，从每个转化体回收的至少一部分质粒DNA携带功能性APHII基因，这表明附加型载体DNA可能在维持对G-418的抗性表型中发挥了作用。
