Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis

Dwight H. Hall, Christine M. Povinelli, Karen Ehrenman, Joan Pedersen-Lane, Frederick Chu, Marlene Belfort

Research output: Contribution to journalArticlepeer-review

Abstract

Of 97 nondirected T4 thymidylate synthase-defective (td) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5′ domain and tdN47 destabilizing the P9 helix in the 3′ domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4tdN57 and T4tdN47, both of which are impaired in cleavage at the 5′ and 3′ splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.

Original languageEnglish (US)
Pages (from-to)63-71
Number of pages9
JournalCell
Volume48
Issue number1
DOIs
StatePublished - Jan 16 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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