Ruohola H, Baker S M, Parker R, Platt T
Department of Biochemistry, University of Rochester Medical Center, NY 14642.
Proc Natl Acad Sci U S A. 1988 Jul;85(14):5041-5. doi: 10.1073/pnas.85.14.5041.
We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYC1 mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYC1 fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation of this fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate less than 40% of the control level of full-length mRNA and reveal the presence of a short, stable (approximately equal to 300 nucleotides) poly(A) RNA that represents 60-70% of the transcripts originating from the same promoter. The reverse orientation of the insert allows near-normal levels of full-length mRNA. Mapping of the 3' end of the truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYC1 transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.
我们已将一段跨越酿酒酵母CYC1 mRNA正常3'末端形成位点的82个碱基对区域克隆到一个整合载体中，该载体带有与HIS4ABC序列读框融合的肌动蛋白基因5'末端（包括其内含子）。如果HIS4C（编码组氨酸醇脱氢酶）充分表达，此载体可赋予在组氨酸醇上生长的能力。当CYC1片段以其野生型（正向）方向克隆到肌动蛋白内含子中时，转化体无法在组氨酸醇上生长，而用携带该片段反向方向的载体转化的细胞则能够良好生长。RNA印迹表明，含有正向方向的转化体积累的全长mRNA不到对照水平的40%，并揭示存在一种短的、稳定的（约300个核苷酸）多聚腺苷酸化RNA，其占来自同一启动子转录本的60 - 70%。插入片段的反向方向允许全长mRNA接近正常水平。对截短RNA的3'末端进行定位表明，多聚腺苷酸化的长度可变，但发生在与正常CYC1转录本相同的位置。显性和隐性抑制突变使转化体尽管有插入片段仍能在组氨酸醇上生长。遗传分析表明，大多数显性突变体是顺式作用的，隐性突变体至少定义了三个互补组，这表明几个不同基因的缺陷可恢复更高水平的HIS4C表达。
