Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.
Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.
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使用上游启动子元件和保守基因内区域特异的引物扩增植物 U3 和 U6 snRNA 基因序列。
DOI：
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发表时间：
1990
影响因子：
14.9
通讯作者：
Witold Filipowicz
中科院分区：
文献类型：
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作者：
C. Marshallsay;Tamás Kiss;Witold Filipowicz
高等植物中的U-snRNA基因包含两个基本的启动子元件，具有序列RTCCCACATCG的USE和TATA样盒，分别位于-70和-30区域。使用含有USE基序的寡脱氧核苷酸和对U-snRNA中基因内保守区域特异的寡脱氧核苷酸，通过聚合酶链反应（PCR）扩增拟南芥和烟草基因组DNA来确定编码U6和U3 snRNA的几个序列。该方法为植物U-snRNA基因及其启动子的鉴定提供了一种简单、快速的方法。它也可以用于表征其他基因含有保守的上游启动子元件。PCR衍生的片段被用作探针，用于从拟南芥基因组文库中分离U3 snRNA基因。两个分离的U3基因被证明是活跃的，当转染到烟草plumbaginifolia原生质体。两个U3基因都含有USE和TATA样上游元件，位于与拟南芥的U6基因相似的位置。编码的拟南芥U3 snRNA可以折叠成二级结构，其更类似于来自低等真核生物而不是来自后生动物的U3 RNA。
U-snRNA genes in higher plants contain two essential promoter elements, the USE with sequence RTCCCACATCG and the TATA-like box, positioned in the -70 and -30 regions, respectively. Using an oligodeoxynucleotide containing the USE motif and oligodeoxynucleotides specific for the intragenic regions conserved in U-snRNAs, several sequences encoding U6 and U3 snRNAs were determined by polymerase chain reaction (PCR) amplification of Arabidopsis thaliana and tobacco genomic DNAs. This method provides a simple and rapid procedure for characterisation of plant U-snRNA genes and their promoters. It could also be used for the characterisation of other genes containing conserved upstream promoter elements. PCR-derived fragments were used as probes for the isolation of the U3 snRNA genes from a genomic library of Arabidopsis. Two isolated U3 genes were shown to be active when transfected into protoplasts of Nicotiana plumbaginifolia. Both U3 genes contain the USE and TATA-like upstream elements located in similar positions to the U6 genes of Arabidopsis. The encoded Arabidopsis U3 snRNAs can be folded into a secondary structure which is more similar to that of U3 RNAs from lower eukaryotes rather than from metazoa.