Sizova I A, Lapina T V, Frolova O N, Alexandrova N N, Akopiants K E, Danilenko V N
Biological Research Institute, St. Petersburg State University, Russia.
Gene. 1996 Nov 28;181(1-2):13-8. doi: 10.1016/s0378-1119(96)00384-8.
The aminoglycoside 3'-phosphotransferase type VIII (APHVIII) encoding gene (aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7-8 mt+ for paromomycin resistance (PmR) at a frequency (1.3-1.9) x 10(-7), probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The PmR phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.
通过玻璃珠高效转化法，将来自龟裂链霉菌的氨基糖苷3'-磷酸转移酶VIII型（APHVIII）编码基因（aphVIII）导入绿色单细胞藻类莱茵衣藻的核基因组中，以诱导对氨基糖苷类抗生素具有抗性的转化体。aphVIII结构序列两侧是龟裂链霉菌的调控序列，但这些序列未能在莱茵衣藻中指导表达。含有这些序列的pSU937质粒能够以（1.3 - 1.9）×10⁻⁷的频率转化莱茵衣藻cw15 arg7 - 8 mt⁺菌株，使其获得巴龙霉素抗性（PmR），这可能是由于体内基因融合以及aphVIII基因从核DNA调控元件表达的结果。莱茵衣藻真正发生转化的证据包括用针对aphVIII的特异性探针进行印迹杂交，以及在转化体的粗细胞提取物中证明APHVIII酶活性。在有选择和无选择的情况下，整合的链霉菌DNA序列、APHVIII酶活性和氨基糖苷抗性表型在有丝分裂过程中都是稳定的。PmR表型可由转化体与野生型菌株杂交产生的减数分裂后代遗传。
