The tRNA(Ser)-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and...

The existence of six serine codons results in a complex pattern of tRNA(Ser) isoacceptors in organisms and organelles. According to the original wobble hypothesis, a minimum of three isoacceptors should be sufficient to read the six serine codons. We have isolated five cytoplasmic tRNAs(Ser) from leaves of Nicotiana rustica. Their nucleotide sequences identify them as four different isoacceptors with the anticodons cm5UGA, CGA, IGA and GCU. For tRNA(Ser) with IGA anticodon, two species have been detected which vary only by one nucleotide in the long extra arm. The first three isoacceptors recognize codons of the type UCN whereas the fourth isoacceptor reads the two serine codons AGC and AGU. The tRNA(Ser) sequences were used to design appropriate primers for the amplification of Nicotiana nuclear tRNA(Ser) genes by the polymerase chain reaction (PCR). A total number of eight tRNA(Ser) genes differing in the coding region were thus identified. Selected PCR DNA fragments were then employed as probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. Sequence analyses revealed that five of the isolated seven clones contained tRNA(Ser) genes which are identical in sequence with the five cytoplasmic tRNAs(Ser) mentioned above. None of them contains an intervening sequence. This 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 plant extract. The approximate tRNA(Ser) gene number was estimated by hybridization of specific DNA probes to Eco RI-cleaved Nicotiana nuclear DNA. The overall hybridization pattern indicates that members of each particular tRNA(Ser) gene family do not appear to be clustered but distributed randomly throughout the Nicotiana genome.