Randolph-Anderson B L, Boynton J E, Gillham N W, Harris E H, Johnson A M, Dorthu M P, Matagne R F
Department of Botany, Duke University, Durham, NC 27706.
Mol Gen Genet. 1993 Jan;236(2-3):235-44. doi: 10.1007/BF00277118.
The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.
莱茵衣藻的呼吸缺陷型dum-1突变体由于线性15.8 kb线粒体基因组末端有1.5 kb的缺失，影响了脱辅基细胞色素b（CYB）基因，因而无法在黑暗中生长。与仅观察到单体长度线粒体基因组的野生型不同，dum-1基因组以单体和二聚体长度分子的混合物形式存在。突变体二聚体似乎是由两个缺失分子的头对头融合产生的。此外，还发现dum-1的线粒体基因组不稳定，原始突变群体中单个细胞克隆的缺失程度各不相同。dum-1突变体每代还会以约4%的频率分离出致死微小菌落，其中原始缺失现在至少延伸到相邻的编码NAD脱氢酶亚基四（ND4）的基因中。我们以dum-1突变体为受体，采用生物弹道法在莱茵衣藻中证明了线粒体的稳定转化。经过4至8周的黑暗培养，从用莱茵衣藻线粒体DNA轰击的dum-1细胞平板上共分离出22个呼吸功能正常的菌落（频率为7.3×10^(-7)），从用史密斯衣藻线粒体DNA轰击的平板上分离出1个菌落（频率为0.8×10^(-7)）。对照平板上未观察到菌落（频率<0.96×10^(-9)）。所有转化体在黑暗中于乙酸盐培养基上正常生长；22个转化体对于莱茵衣藻供体典型的野生型线粒体基因组是同质性的。从史密斯衣藻供体获得的单个转化体具有重组线粒体基因组，其中包含供体CYB基因以及来自莱茵衣藻受体的细胞色素氧化酶亚基I（COI）编码基因中的诊断性HpaI和XbaI限制性位点。在任何转化体中均未检测到dum-1受体的特征性缺失片段。
