Nucleic Acids Research, 1988, Vol. 16, No. 24 11591-11606
© 1988
MOLECULAR BIOLOGY |
Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expresanon in vitro and in vivo
Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, USA
Received August 9, 1988. Revised November 17, 1988. Accepted November 17, 1988.
The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was introduced into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to 
Total tRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.