Mason P J, Jones M B, Elkington J A, Williams J G
EMBO J. 1985 Jan;4(1):205-11. doi: 10.1002/j.1460-2075.1985.tb02337.x.
We show that adult reticulocytes of Xenopus laevis produce two forms of the beta 1 globin mRNA that differ in their site of polyadenylation. The minor site of polyadenylation is located 46 nucleotides downstream of the major site and is used in approximately 1% of mRNA molecules. A fusion gene was constructed containing the promoter from the thymidine kinase gene of herpes simplex virus fused to the protein coding, 3'-noncoding and 3'-flanking sequences of the X. laevis beta 1 globin gene. When injected into the nuclei of Xenopus oocytes, transcripts of this fusion gene were accurately and efficiently spliced and polyadenylated. The proportion of fusion gene transcripts terminating at the major and minor polyadenylation sites after injection into oocytes was approximately similar to that found in reticulocytes. When the AATAAA sequence element upstream from the major site was deleted, the minor site was used with a high (greater than 90%) efficiency. Therefore, by comparing the ratio of polyadenylation at the major and minor sites, it is possible to determine the effect of sequence alterations at the major site. In a construct where the AATAAA polyadenylation signal was changed to AATACA a high proportion (35%) of transcripts continued to be polyadenylated at the major site. This suggests a surprisingly high degree of flexibility in the precise polyadenylation signal.
我们发现非洲爪蟾的成年网织红细胞产生两种形式的β1珠蛋白mRNA，它们的多聚腺苷酸化位点不同。次要的多聚腺苷酸化位点位于主要位点下游46个核苷酸处，约1%的mRNA分子使用该位点。构建了一个融合基因，其包含单纯疱疹病毒胸苷激酶基因的启动子，与非洲爪蟾β1珠蛋白基因的蛋白质编码、3'非编码和3'侧翼序列融合。当将该融合基因的转录本注射到非洲爪蟾卵母细胞核中时，它们能被准确且高效地剪接和多聚腺苷酸化。注射到卵母细胞后，在主要和次要多聚腺苷酸化位点终止的融合基因转录本的比例与在网织红细胞中发现的比例大致相似。当主要位点上游的AATAAA序列元件被删除时，次要位点的使用效率很高（大于90%）。因此，通过比较主要和次要位点的多聚腺苷酸化比例，可以确定主要位点序列改变的影响。在一个将AATAAA多聚腺苷酸化信号改变为AATACA的构建体中，相当比例（35%）的转录本继续在主要位点进行多聚腺苷酸化。这表明精确的多聚腺苷酸化信号具有惊人的高度灵活性。
