FURTHER CHARACTERIZATION OF THE RESPIRATORY DEFICIENT DUM-1 MUTATION OF CHLAMYDOMONAS-REINHARDTII AND ITS USE AS A RECIPIENT FOR MITOCHONDRIAL TRANSFORMATION
FURTHER CHARACTERIZATION OF THE RESPIRATORY DEFICIENT DUM-1 MUTATION OF CHLAMYDOMONAS-REINHARDTII AND ITS USE AS A RECIPIENT FOR MITOCHONDRIAL TRANSFORMATION
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DOI：
10.1007/bf00277118
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发表时间：
1993-01-01
期刊：
影响因子：
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通讯作者：
MATAGNE, RF
中科院分区：
文献类型：
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作者：
RANDOLPHANDERSON, BL;BOYNTON, JE;MATAGNE, RF
莱茵衣藻DUM-1呼吸缺陷突变体在15.8kb的线粒体基因组中存在1.5kb的末端缺失，影响了细胞色素b(Cyb)基因的表达，使其不能在黑暗中生长。与只观察到单体长度的线粒体基因组的野生型不同，DUM-1基因组是单体和二聚体长度分子的混合物。突变的二聚体似乎是两个缺失分子头对头融合的结果。此外，DUM-1的线粒体基因组也是不稳定的，缺失的程度在原始突变群体的单细胞克隆中有所不同。DUM-1突变体还以每代约4%的频率分离致死微小菌落，其中原始缺失现在至少延伸到编码NAD脱氢酶(ND4)亚单位4的相邻基因。我们以DUM-1突变体为受体，用生物学方法证明了线粒体在莱茵梭菌中的稳定转化。暗培养4~8周后，从雷氏弧菌线粒体DNA轰击的DUM-1细胞平板中分离到22个呼吸活性菌落(频率7.3×10(-7))，从史密斯弧菌线粒体DNA轰击的平板中分离到单个菌落(频率0.8×10(-7))。对照平板上未见菌落(频率<lt；0.96×10(-9))。所有转化子都能在醋酸介质中的黑暗中正常生长；22个转化子的野生型线粒体基因组是典型的莱茵哈迪尔乳杆菌供体的同质。从供体获得的单个转化子含有重组线粒体基因组，其中含有供体的CyB基因，以及来自受体的细胞色素氧化酶(COI)亚基I基因的HPAI和XbaI限制性内切酶切点。在任何转化子中都没有检测到DUM-1受体的特征缺失片段。
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.