Orias E, Larson D, Hu Y F, Yu G L, Karttunen J, Løvlie A, Haller B, Blackburn E H
Department of Biological Sciences, University of California, Santa Barbara 93106.
Gene. 1988 Oct 30;70(2):295-301. doi: 10.1016/0378-1119(88)90201-6.
The macronucleus of the ciliate Tetrahymena contains approx. 10(4) ribosomal RNA gene molecules (rDNA) in the form of linear, autonomously replicating palindromes. Previous studies have shown that macronuclear rDNA molecules derived from wild-type (wt) inbred strain C3 out-replicate those derived from wt inbred strain B, in macronuclei initially heterozygous for both, leading to the complete loss of the B rDNA. However, rmm-1, a cis-acting laboratory-induced mutation obtained previously by mutagenesis of inbred strain C3, causes the mutant rmm-1 rDNA to be completely out-replicated by B rDNA. These findings suggest the following hierarchy of replication potential: wt C3 greater than wt B greater than C3-rmm-1. We used electroporation to test whether cells containing only rmm-1 macronuclear rDNA are favorable recipients for transformation with either wt B or C3 donor rDNA molecules. The donor rDNA molecules carried the selectable marker Pmr (paromomycin resistance) located in the coding region of the 17S rRNA. Transformants were obtained, at a frequency greater than 1 in 10(5), by electroporation under a wide range of electrical discharge parameters. The fraction of cells surviving electroporation varied between 2 and greater than 95% in successful experiments. Replacement ('transplacement') of the recipient rDNA was observed, consistent with the prediction that B and C3 rDNA should out-replicate rmm-1 rDNA. These findings are also consistent with the previous conclusion that the differential replication determinants reside in the 5'-nontranscribed spacer of the rDNA.
纤毛虫四膜虫的大核含有约10⁴个核糖体RNA基因分子（rDNA），呈线性、自主复制的回文结构形式。先前的研究表明，在最初对野生型（wt）近交系C3和wt近交系B均杂合的大核中，源自野生型近交系C3的大核rDNA分子比源自wt近交系B的大核rDNA分子复制得更快，导致B rDNA完全丢失。然而，rmm - 1是先前通过对近交系C3进行诱变获得的一种顺式作用的实验室诱导突变，它导致突变的rmm - 1 rDNA被B rDNA完全超越复制。这些发现表明了以下复制潜力等级：wt C3＞wt B＞C3 - rmm - 1。我们使用电穿孔来测试仅含有rmm - 1大核rDNA的细胞是否是用wt B或C3供体rDNA分子进行转化的合适受体。供体rDNA分子携带位于17S rRNA编码区的可选择标记Pmr（巴龙霉素抗性）。在广泛的放电参数下通过电穿孔获得了频率大于1/10⁵的转化体。在成功的实验中，电穿孔后存活的细胞比例在2%至大于95%之间变化。观察到受体rDNA的替代（“转位替代”），这与B和C3 rDNA应该超越rmm - 1 rDNA复制的预测一致。这些发现也与先前的结论一致，即差异复制决定因素存在于rDNA的5' - 非转录间隔区。
