MOLECULAR CHARACTERIZATION OF STREPTOMYCIN RESISTANCE AND THE TRANS-SPLICING RPS12 GENE IN NICOTIANA TABACUM CHLOROPLASTS.

Abstract

Streptomycin resistance in E. coli ribosomes is conferred by alterations in the amino acid sequence of 30S ribosomal protein S12. The alterations result from point mutations at specific locations in the rps12 gene. A point mutation at a conserved nucleotide in the 16S rRNA gene, originally identified in Euglena gracilis chloroplasts, also confers streptomycin resistance to prokaryotic-like ribosomes. The Nicotiana tabacum mutant "SR1" possesses a chloroplast-linked streptomycin resistance allele. The results presented in this thesis identify a mutation in SR116S rRNA, which occurs at the same position as in streptomycin resistant Euglena mutants. The tobacco chloroplast rps12 gene has been characterized. This gene is expressed in a unique way; two separate transcripts encoding different portions of the gene undergo a bimolecular (trans-) splicing event during mRNA maturation. C-terminal rps12 exons 2 and 3 were identified in the inverted repeat regions of the tobacco chloroplast genome. Complementary DNA sequencing of mature rps12 mRNA allowed deduction of the remaining N-terminal (exon 1) sequence. Hybridizations with synthetic oligodeoxyribonucleotide primers complementary to the deduced RNA sequence located the coding region of exon 1 to be 29 kilobasepairs (kbp) downstream of the nearest copy, and 69 kbp away from, and on the opposite DNA strand of, the distal copy, of exons 2 and 3. Northern hybridization analysis and primer extension sequencing of cDNA of rps12 transcripts indicate that exon 1 and exons 2-3 are encoded on separate transcripts. Exon 1 and exons 2-3 are covalently ligated in mature rps12 mRNA. Therefore, the separate transcripts encoding exon 1 and exons 2-3 undergo a trans-splicing event during the maturation of rps12 mRNA. A complete cloned library of tobacco chloroplast DNA was obtained, consisting of overlapping Bam HI restriction fragments. Three new restriction maps of tobacco chloroplast DNA, for the enzymes Sma I, Kpn I, and Bam HI, were derived by two-dimensional gel analysis and a novel computer-aided mapping technique.