Suyama Y, Fukuhara H, Sor F
Department of Biology, University of Pennsylvania, Philadelphia 19104.
Curr Genet. 1985;9(6):479-93. doi: 10.1007/BF00434053.
A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis strain ST is presented. 1. Based on agarose gel electrophoresis data together with limited nucleotide sequences available on some restriction fragments, we estimate the actual size of this genome to be about 55,000 base pairs. 2. Seven tRNA gene locations have been assigned, which are scattered along the genome length. Six of these locations encode the genes for tRNA(phe), tRNA(his), tRNA(trp), and tRNA(glu), and the duplicate tRNA(tyr) genes which are located at the inverted terminal repeat segments. The tRNA gene(s) encoded in one location has not been identified. We have not yet found the tRNA(leu) and tRNA(met) genes, which were previously shown to be encoded in the genome (Chiu et al. 1974; Suyama 1982). 3. We have mapped the 14S rRNA gene by sequencing the 170 bp segment of EcoRI fragment 8 and by aligning its sequence with E. coli 16S rRNA. From our recent complete sequence data the gene size was found to be about 1,650 bp, which is unexpectedly large for the 14S rRNA which has an estimated size of 1,300 bp. The 14S rRNA is probably a cleavage product of the larger primary transcript of which 200-300 bases of the 5' end are missing. 4. The duplicate copies of the 21S rRNA gene at the terminal duplication inversion segments were analyzed. ClaI fragment 7 (1,500 bp) corresponds in sequence from base position 850 to 2,390 of the 20S rRNA gene of Paramecium mitochondrial DNA (Seilhamer et al. 1984b). The 21S gene is approximately 2,500 bp long. The presence of some restriction site polymorphism is apparent in this segment. 5. Each of the 21S gene copies precedes the tRNA(tyr) gene, but the space flanking one tRNA(tyr) gene differs in size and restriction sites from the space flanking another tRNA(tyr) gene. Thus, this space corresponds to the segment of an imperfect match in the terminal duplication inversion of Goldbach et al. (1978a). 6. Saccharomyces cerevisiae mitochondrial probes including Cob, ATPase VI and IX, and cytochrome oxidase I gene sequences, 21S and 15S rRNAs, and mouse mitochondrial DNA showed no significant hybridization with any restriction fragments of Tetrahymena mitochondrial DNA. The results are in accordance with an extensive sequence divergence previously found in the Tetrahymena mitochondrial genome (Goldbach et al. 1977).
本文给出了梨形四膜虫ST株系线性线粒体DNA的精细限制酶切图谱。1. 根据琼脂糖凝胶电泳数据以及部分限制片段上有限的核苷酸序列，我们估计该基因组的实际大小约为55,000个碱基对。2. 已确定了7个tRNA基因的位置，它们沿基因组长度分散分布。其中6个位置编码tRNA（苯丙氨酸）、tRNA（组氨酸）、tRNA（色氨酸）和tRNA（谷氨酸）的基因，以及位于反向末端重复序列段的重复tRNA（酪氨酸）基因。位于一个位置的tRNA基因尚未确定。我们尚未找到tRNA（亮氨酸）和tRNA（甲硫氨酸）基因，它们先前已被证明是在基因组中编码的（Chiu等人，1974年；Suyama，1982年）。3. 通过对EcoRI片段8的170 bp片段进行测序，并将其序列与大肠杆菌16S rRNA进行比对，我们绘制了14S rRNA基因图谱。从我们最近的完整序列数据中发现该基因大小约为1,650 bp，对于估计大小为1,300 bp的14S rRNA来说，这个大小出乎意料地大。14S rRNA可能是较大初级转录本的切割产物，其5'端缺失200 - 300个碱基。4. 分析了末端重复倒位片段处21S rRNA基因的重复拷贝。ClaI片段7（1,500 bp）与草履虫线粒体DNA的20S rRNA基因从第850位到2,390位的序列相对应（Seilhamer等人，1984b）。21S基因大约长2,500 bp。在该片段中明显存在一些限制酶切位点多态性。5. 每个21S基因拷贝都位于tRNA（酪氨酸）基因之前，但一个tRNA（酪氨酸）基因两侧的间隔在大小和限制酶切位点上与另一个tRNA（酪氨酸）基因两侧的间隔不同。因此，这个间隔对应于Goldbach等人（1978a）末端重复倒位中不完全匹配的片段。6. 酿酒酵母线粒体探针，包括Cob、ATPase VI和IX以及细胞色素氧化酶I基因序列、21S和15S rRNA，以及小鼠线粒体DNA，与梨形四膜虫线粒体DNA的任何限制片段均未显示出明显的杂交信号。这些结果与先前在梨形四膜虫线粒体基因组中发现的广泛序列差异一致（Goldbach等人，1977年）。
