UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coil genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNA"rP gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.We recently have sequenced a part of the Mycoplasma capricolum ribosomal-protein gene cluster that codes for polypeptides highly homologous to the Escherichia coli ribosomal proteins S3 and L16. The sequence contains four UGA codons in the reading frames; three appear at the sites corresponding to tryptophan, and one, at a site corresponding to arginine in the E. coli proteins. No "universal" UGG codon for tryptophan has so far been found. We have also isolated a clone containing a pair of M. capricolum tRNA genes, the sequence of both of which resembles that of tRNATrp of E. coli. The anticodon sequence of one of these tRNA genes is 5'-T-C-A-3', which can base-pair with both opal codon UGA and universal tryptophan codon UGG. That of the other is 5'-C-C-A-3', which may base-pair exclusively with UGG. These two tRNA genes are expressed in the cell. All these findings suggest strongly that, in M. capricolum, UGA codes for tryptophan using the opal tRNAUCA but not tRNACCA.RESULTS AND DISCUSSION UGA Codons in M. capricolum S3 and L16 Genes. As reported in a previous paper (1), we isolated the recombinant plasmid pMCB1088 containing a 9-kilobase-pair fragment of M. capricolum DNA. The fragment contains the genes for at least nine ribosomal proteins-S3, S5, S8, S14, S17, L5, L6, L16, and L18-as deduced from its encoded protein sequences being highly homologous with the corresponding E. coli ribosomal protein sequences (refs. 1 and 2; unpublished results). Fig. 1 shows the complete nucleotide sequence of a 629-base-pair (bp) HindIII fragment which is a part of the insert of pMCB1088 (see refs. 1 and 2). The DNA corresponds to the 3' half of the S3 gene and about 90% of the L16 gene from the 5' terminus. When the M. capricolum sequences are aligned with the E. coli protein sequences (3, 4) ( Fig. 1), four UGA (opal) codons are found within the reading frames. The possibility that these UGA codons are termination signals can be excluded by their occurrence in the regions having extensive sequence homologies with the E. coli proteins. More importantly, three out of the four UGA codons appear at the positions corresponding to tryptophan in the E. coli proteins. This suggests that UGA is a sense codon, probably for tryptophan, in M. capricolum. No UGG codon for tryptop...
Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2D mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2D mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damageinduced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5D and rhp57D mutants and completely abolished in an swi5D rhp57D double mutant. An assay for products generated by HO endonucleaseinduced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.
The DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined. The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli. The deduced amino acid sequence of each protein is also well conserved in the two bacteria. The G + C content of the M. capricolum genes is 29%, which is much lower than that of E. coli (51%). The codon usage pattern of M. capricolum is different from that of E. coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position. UGA, which is a stop codon in the "universal" code, is used more abundantly than UGG to dictate tryptophan.
The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNASerUGA tRNA gene is followed by a sequence coding for the initiator tRNAMet. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.