THE TRNA(SER)-ISOACCEPTORS AND THEIR GENES IN NICOTIANA-RUSTICA - GENOME ORGANIZATION, EXPRESSION IN-VITRO AND SEQUENCE ANALYSES

The existence of six serine codons results in a complex pattern of tRN A(Ser) isoacceptors in organisms and organelles. According to the orig inal wobble hypothesis, a minimum of three isoacceptors should be suff icient to read the six serine codons. We have isolated five cytoplasmi c tRNAs(Ser) from leaves of Nicotiana rustica. Their nucleotide sequen ces;identify them as four different isoacceptors with the anticodons c m(5)UGA, CGA, IGA and GCU. For tRNA(Ser) with IGA anticodon, two speci es have been detected which vary only by one nucleotide in the long ex tra arm. The first three isoacceptors recognize codons of the type UCN whereas the fourth isoacceptor reads the two serine codons AGC and AG U. The tRNA(Ser) sequences were used to design appropriate primers for the amplification of Nicotiana nuclear tRNA(Ser) genes by the polymer ase chain reaction (PCR). A total number of eight tRNA(Ser) genes diff ering in the coding region were thus identified. Selected PCR DNA frag ments were then employed as probes for the isolation of the correspond ing genes from a nuclear DNA library of N. rustica. Sequence analyses revealed that five of the isolated seven clones contained tRNA(Ser) ge nes which are identical in sequence with the five cytoplasmic tRNAs(Se r) mentioned above. None of them contains an intervening sequence. Thi s is the first time that all putative cellular tRNA(Ser) isoacceptors and their corresponding genes have been characterized in an eukaryotic organism. Most of the tRNA(Ser) genes are functional as deduced from in vitro transcription and processing studies. Two of the genes yield pre-tRNAs(Ser) which are not or poorly converted to mature tRNA in a p lant extract. The approximate tRNA(Ser) gene number was estimated by h ybridization of specific DNA probes to Eco RI-cleaved Nicotiana nuclea r DNA. The overall hybridization pattern indicates that members of eac h particular tRNA(Ser) gene family do not appear to be clustered but d istributed randomly throughout the Nicotiana genome.