Jacquier A, Rodriguez J R, Rosbash M
Cell. 1985 Dec;43(2 Pt 1):423-30. doi: 10.1016/0092-8674(85)90172-2.
We have introduced four point mutations into the 5' junction (GTATGT) and TACTAAC box of a yeast intron-containing gene coding for beta-galactosidase. To analyze quantitatively mutant combinations, we deliberately avoided nucleotides adjacent to the sites of cleavage (GTATGT) and lariat formation (TACTAAC) and chose positions with expected weak effects on splicing. All four mutants do not affect, or only marginally affect, gene expression. The levels of precursor RNA and intermediates indicate, however, that splicing efficiency is affected in all cases; the first stage of splicing, 5' cleavage and lariat formation, is reduced two to 15-fold in these four mutants. The mutants were combined to generate compensatory and noncompensatory double mutant combinations. No evidence for a specific base-pairing interaction between the 5' junction and TACTAAC box could be obtained. The results suggest that all conserved positions in the 5' junction and TACTAAC box play a role in splicing efficiency.
我们已将四个点突变引入编码β-半乳糖苷酶的含酵母内含子基因的5'连接点（GTATGT）和TACTAAC框。为了定量分析突变体组合，我们特意避开了与切割位点（GTATGT）和套索形成位点（TACTAAC）相邻的核苷酸，并选择了对剪接预期影响较弱的位置。所有四个突变体均不影响或仅轻微影响基因表达。然而，前体RNA和中间体的水平表明，在所有情况下剪接效率均受到影响；在这四个突变体中，剪接的第一阶段，即5'切割和套索形成，降低了2至15倍。将这些突变体组合以产生补偿性和非补偿性双突变体组合。未获得5'连接点与TACTAAC框之间存在特异性碱基配对相互作用的证据。结果表明，5'连接点和TACTAAC框中的所有保守位置在剪接效率中均起作用。
