Intron bypass: a rapid procedure for eliminating introns from cloned genomic DNA and its application to a cellulase gene.
We have devised a DNA cloning procedure in which the introns present in a genomic DNA fragment can be eliminated easily and rapidly. The technique combines the methods of cDNA and genomic cloning in a way which assures full-length representation of the intron-free transcript. Moreover, plasmids made by this technique can be designed to contain flanking untranscribed regions which may play a role in the regulation of expression. One strand of a linearized plasmid containing the 3'-end of a gene is used to prime cDNA synthesis from an annealed mRNA template. A second plasmid containing the 5'-end of the gene is linearized, denatured, and annealed to the extended 3'-end molecules and the resulting circular, partial duplexes are used to transform bacterial cells. Two different recombinant plasmids which contain DNA encoding the cellulase, exocellobiohydrolase I, from Trichoderma reesei have been constructed using this method. They both contain the entire translated region of the gene uninterrupted by introns. One plasmid contains additional DNA at the 5'-end, including approximately 150 bp 5' to the start of transcription. The inserts of both plasmids can be excised in one piece.
D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D003904:Mitosporic Fungi / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D014170:Transformation, Genetic; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
D004274:DNA, Recombinant; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D002480:Cellulase