Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.
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.
Marshallsay
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C
Friedrich Miescher-Institut, Basel, Switzerland.
Kiss
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Filipowicz
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D001483:Base Sequence; D005786:Gene Expression Regulation; D017343:Genes, Plant; D007438:Introns; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010944:Plants / Q000235:genetics*; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012342:RNA, Small Nuclear / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014026:Nicotiana / Q000235:genetics*
D012342:RNA, Small Nuclear