Mol Gen Genet (1982) 187:236-239 © Springer-Verlag 1982 Altered Genetic Code in Paramecium Mitochondria: Possible Evolutionary Trends Jeffrey J. Seilhamer and Donald J. Cummings Department of Microbiology and Immunology, B-175, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA Summary. The sequence and presumptive structure of a tRNA trp gene from Paramecium tetraaurelia are given. The gene is located 1,500 bp downstream from the 13S rRNA gene, in about the middle of the genome. Parame- cium tRNA trp has a completely normal T~uC loop and stem, however its anticodon (UCA) constitutes an alter- ation in the "universal" genetic code, similar to those seen in fungal and mammalian mitochondria. Most features of Paramecium tRNA trp resemble other mitochondrial coun- terparts; however, its sequence is more homologous to the "unaltered" tRNA trp (anticodon CCA) from E. coli. Paramecium mitochondria may resemble a primitive stage of organelle evolution. Introduction Deviations in the "universal" genetic code have been de- tected in mitochondria of mammals (Anderson et al. 1981 ; Barrell et al. 1979; Barrell et al. 1980; Bibb et al. 1981; Young and Anderson 1980) and fungi (Bonitz et al. 1980; Fox 1979; Heckman et al. 1980; Li and Tzagoloff 1979; Macino et al. 1979; Mahler 1981; Sibler et al. 1981). Of these deviations, some are common to both groups, while others are not. In particular, both mammalian and fungal mitochondria contain a tRNA specific for tryptophan which carries the anticodon UCA, complementary to the opal terminator codon (UGA) of the "universal" code. On the other hand, CUN codons (which specify leucine in the universal code) code for threonine in yeast mitochon- dria exclusively (Heckman et al. 1980; Li and Tzagoloff 1979; Sibler et al. 1981). Also, the codon AUA, normally specifying isoleucine, codes for formylmethionine in human mitochondria (Barrell et al. 1979; Barrell et al. 1980). In addition, mammalian mitochondrial AGR codons are chain terminators instead of coding for arginine (Barrell et al. 1979; Anderson et al. 1981). Clearly there is no one unique set of code alterations to be found in mitochondria of all organisms. It is therefore of interest to examine other organ- isms for the presence or absence of these alterations. We are currently examining the structure and sequence of various mitochondrial genes of Paramecium. Parame- cium represents a third distinct animal Kingdom. Further, its genome, a 14 g linear molecule which replicates unidirec- tionally from one unique end (Goddard and Cummings 1977; Pritchard and Cummings 1981), is quite distinct from mammals and fungi. We report here the sequence of the presumptive tRNA trp gene from Paramecium which con- tains the opal to trp alteration seen as well in both mammals and fungi. In addition, the evolution of tRNA trp genes and implications for organelle evolution are considered. Materials and Methods Restriction fragments containing the large, small, and flanking sequences of the ribosomal RNA genes from mito- chondrial DNA of Paramecium tetraaurelia, stock 51s, were cloned into pBR325 by Jane Laping in our laboratory. Clonal DNA was end-labelled, midcut, separated, and se- quenced by the technique of Maxam and Gilbert (1977). The tRNA trp gene was located by scanning the completed sequence for the T~uC stem and loop. Comparisons of cloned and native genomic Paramecium mitochondrial DNAs have failed to detect any differences due to cloning. Results and Discussion Paramecium contains one copy each of the two rRNA genes, the large (20s) located near the replicative terminal end and the small (13S) near the middle of the genome (Cummings et al. 1980; Cummings and Laping 1981). We are currently sequencing both genes and their flanking regions in the process of studying gene organization and expression. Through computer analysis, we identified a tRNA gene about 1500 base pairs downstream from the 3' end of the P. tetraaurelia 13S rRNA gene. The tRNA gene is transcribed from the same strand as are the other currently identified genes. The sequence and structure of the presumptive tRNA as deduced from the DNA sequence is shown in Fig. 1, along with 45 bases of flanking sequence. The structure contains no base pairing mismatches except for the presence of single G: U pairings in three of the stems. Note that, as with most eucaryotic tRNAs, the 3'-ter- minal CCA sequence is not present within the DNA se- quence, but presumably is added posttransmiptionally. We have not yet demonstrated expression of this gene; however, its high degree of structural integrity, strand location and homology (below) suggest strongly that it is indeed func- tional in vivo. The anticodon present within this tRNA, UCA, is of immediate significance, since it is able to base-pair with both the "opal" codon UGA and the normal tryptophan codon UGG by third position G:U wobble (Crick 1966). 0026-8925/82/0187/0236/$01.00
