BMC Genomics 2005, 6:125 http://www.biomedcentral.com/1471-2164/6/125 to a few groups of eubacteria including α-, β-, and γ-pro- teobacteria, actinobacteria, some fungi, and animal spe- cies spanning the phylogenetic range from insects to mammals. Among the vertebrate species for which draft genome sequences and/or a large number of EST sequences are available, we were able to identify rTS sequences from all species with the exception of chicken and mouse [see a complete list of deduced amino acid sequences in Additional file 1]. Furthermore, we observed in all vertebrate species with sufficient genome sequences including human, chimpanzee, rat, and fugu fish, that the TYMS-rTS gene pair is part of a large conserved gene syn- teny among vertebrates (data not shown). While the absence of rTS sequence in chicken may be due to the insufficiency of available genome sequences or ESTs, we were puzzled by the failure to retrieve any rTS sequences in mouse, considering the fact that the mouse genome sequence is now fairly complete and its EST data is quite comprehensive. However, we recently obtained prelimi- nary data showing expression of mouse rTS protein and mRNA (data not shown). Another unusual observation is that the rTS gene does not seem to be present in E. coli while the rTS gene is present in another enterobacte- rium,Yersinia pestis. Similarly, although the rTS gene is present in Anopheles gambiae and several other insect spe- cies, but is not found in Drosophila. So far, no rTS gene has been identified in any plant or yeast species despite the fact that complete genome sequences and comprehensive EST sequences are available for a number of species in these lineages. We also find no rTS sequences from Caenorhabditis elegans or other worm genome sequences. The same is true for all archaebacteria and many eubacte- ria lineages. These observations suggest that the rTS gene originated as a bacterial gene which was either horizon- tally transferred into certain animal and fungal lineages, or alternatively, was lost in all the lineages that do not contain rTS genes. An interesting observation is that, while the rTS branches for the animal lineage and the fun- gal lineage show a topology that agrees well with com- monly accepted tree of life for these species, the branches for the bacterial lineages conflict with the commonly accepted tree for the bacterial lineages (Fig. 4). The latter is demonstrated by the fact that species from the same lin- eage do not always group together, while species from dif- ferent bacterial lineages do cluster together in many cases (Fig. 4). For example, one β-proteobactial rTS sequence (rTS_bc) is grouped with α-proteobacterial sequences, while the other two β-proteobacterial sequences cluster with a γ-proteobacterial sequence and the three rTS sequences from the High G+C gram positive bacteria are located on three different branches, which all consist of sequences from multiple groups (Fig. 4A). Although not all nodes for the bacterial groups are supported by a high bootstrap value, many nodes are supported with high confidence. This type of unexpected phylotree shown by the rTS sequences suggests the possibility of horizontal gene transfer among the bacteria species, which is com- mon [19-21]. The fact that most of the bacteria and fungi that have a rTS gene are human and/or animal pathogens (Aspergillus nidulans, Brucella suis, Brucella melitensis, Bur- kholderia cepacia Burkholderia fungorum, Cryptococcus neo- formans, Yersinia pestis, Silicibacter sp), plant pathogens (Agrobacterium tumefaciens, Gibberella zeae, Xanthomonas axonopodis, Xanthomonas campestris., Ustilago maydis, Mag- naporthe grisea), or in one instance a plant symbiont (S. meliloti), adds weight to the hypothesis of horizontal gene transfer events. In the case of plant pathogens, the plants can serve as a mediator between a bacterial donor and an animal acceptor. This may also suggest that the rTS gene is required to create and/or maintain a certain type of host- pathogen relationship. Therefore, a better understanding of the biological function of the rTS gene may provide new insights for disease control related to these bacterial pathogens. In addition to the discovery of this novel mitochondrial isoform of rTS, our report also represents a good illustra- tion of the use of comparative genomic analysis for iden- tifying novel protein isoforms of genes and for improving existing gene predictions. Through this study, we have made a comprehensive collection of rTS orthologous sequences and have made a list of suggested modifica- tions to the existing gene annotation (see detailed modifi- cations in the supplemental materials). In addition, by searching all annotated human genes, we identified a few other genes in which evolutionarily conserved alternative upstream AUG codons exist (similar situation as rTSβ). These genes include SELL [NM_000655], SPCS1 [NM_014041.1], NT5C3 [NM_016489], and SDN1 [NM_014390] (data not shown). Conclusion In summary, through comparative genomic analyses we revealed an unusual phylogenetic distribution of the rTS gene and identified a novel mitochondria isoform of this gene and verified it experimentally. A mitochondrial location of rTS protein and phylogenetic distribution of this gene provide us with new information that will assist in elucidating its function. Methods Identification of an extended N-terminal region for all available rTS genes To search for all available rTS orthologous sequences, we first queried all protein sequences deposited in the NCBI non-redundant protein database (nr) by BLAST search [21]. Among all identified rTSβ orthologous protein sequences, the ones with an N-terminus equivalent to human rTSβ were identified and their corresponding genomic sequences, including sufficient 5'-end upstream Page 8 of 10 (page number not for citation purposes)