Kim D, Raymond G J, Clark S D, Vranka J A, Johnson J D
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Nucleic Acids Res. 1990 Jul 25;18(14):4215-21. doi: 10.1093/nar/18.14.4215.
Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.
在酿酒酵母中从未鉴定出源自tRNATrp基因的天然存在的抑制突变体。使用寡核苷酸定向诱变从克隆的tRNATrp基因产生潜在的赭石型和乳白型抑制子。体外转录分析表明，该基因的赭石型抑制子形式TRPO积累的前体和tRNA数量与亲本相当。乳白型抑制子TRPOP积累的tRNA少4-5倍。这两种基因形式在体外都经过加工和剪接，以产生具有预期碱基序列的tRNA。将改变后的基因亚克隆到酵母载体中，并导入携带各种琥珀型、赭石型和乳白型突变的酵母菌株中。当通过CEN载体导入时，赭石型和乳白型抑制子形式均未显示出抑制活性。从克隆中删除CEN区域会使拷贝数增加到每个细胞10-20个。当将该基因通过此载体导入时，乳白型抑制子形式显示出中等抑制活性，然而，无论基因拷贝数如何，赭石型抑制子形式均未表现出可检测到的生物学活性。对含有高拷贝数克隆的细胞中tRNATrp的稳态水平进行Northern印迹分析，结果显示tRNATrp的丰度增加了20-100%。
