Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions

• Published in: Nucleic acids research 1990-06, Vol.18 (12), p.3459-3466
• Main Authors: Marshallsay, C, Kiss, T, Filipowicz, W
• Format: Article
• Language: English
• Subjects: Arabidopsis thaliana; Base Sequence; Gene Expression Regulation; Genes, Plant; Introns; Metazoa; Molecular Sequence Data; Nicotiana - genetics; Nicotiana plumbaginifolia; Nicotiana tabacum; Nucleic Acid Conformation; Plants - genetics; Plants, Toxic; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Small Nuclear - genetics; Sequence Homology, Nucleic Acid; Templates, Genetic
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/18.12.3459

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.