When the stop codons TGA, TAA, and TAG are found in the second and third reading frames of a protein-encoding gene, they are considered premature stop codons (PSC). Deinococcus radiodurans disproportionately favored TGA more than the other two triplets as a PSC. The TGA triplet was also found more often in noncoding regions and as a stop codon, though the bias was less pronounced. We investigated this phenomenon in 72 bacterial species with widely differing chromosomal GC contents. Although TGA and TAG were compositionally similar, we found a great variation in use of TGA but a very limited range of use of TAG. The frequency of use of TGA in the gene sequences generally increased with the GC content of the chromosome, while the frequency of use of TAG, like that of TAA, was inversely proportional to the GC content of the chromosome. The patterns of use of TAA, TGA and TAG as real stop codons were less biased and less influenced by the GC content of the chromosome. Bacteria with higher chromosomal GC contents often contained fewer PSC trimers in their genes. Phylogenetically related bacteria often exhibited similar PSC ratios. In addition, metabolically versatile bacteria have significantly fewer PSC trimers in their genes. The bias toward TGA but against TAG as a PSC could not be explained either by the preferential usage of specific codons or by the GC contents of individual chromosomes. We proposed that the quantity and the quality of the PSC in the genome might be important in bacterial evolution.
Rationale: evaluation of the T-cell receptor (TCR) V-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V primers instead of the conventionally labeled downstream C primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C primer and an Omniscript RT kit. The 24 V primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: V spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V primers. The resolution was superior to that obtained with the labeled C primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor V-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V repertoire analyses in clinical trials.
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