BMC Genomics 2005, 6:125 http://www.biomedcentral.com/1471-2164/6/125 (rTSα and rTSβ) through alternative RNA splicing [2,3]. The mRNA for rTSα is complementary to thymidylate syn- thase mRNA, while the mRNA for rTSβ is not [2,3]. The rTSβ protein is the major protein product of the rTS gene and its expression is associated with the down-regulation of thymidylate synthase protein as cultured cells enter growth arrest [2]. Expression of rTSβ correlates with the production of small molecules that appear to mediate the down-regulation of thymidylate synthase protein by a novel intercellular signaling mechanism [2]. Overproduc- tion of rTSβ occurs in some cells resistant to inhibitors of thymidylate synthase or dihydrofolate reductase, indicat- ing a role for the rTS gene in folate and nucleotide metab- olism, as well as anticancer drug resistance [2-6]. While the specific function(s) of the rTS gene products are currently under investigation, we now report a new rTS protein isoform and its association with mitochondria. The existence of this new isoform, rTSγ, was first predicted using a computational comparative genomic sequence analysis approach and was then verified experimentally. This unexpected observation suggests that rTS may have functions in addition to intercellular signaling. Results A conserved extended protein N-terminus can be deduced from all available rTS genes Comprehensive analysis of all available database sequences revealed that rTS genes demonstrate an atypical phylogenetic distribution. rTS exists only in a few groups of eubacteria, two fungal lineages (Ascomycota and Basidi- omycota), and most animal species from insects to mam- mals. Among bacterial rTS orthologous genes, several were annotated with a longer N-terminus based on a start codon located further upstream. These proteins include NP_355739.1 (Agrobacterium tumefaciens str. C58), NP_540624.1(Brucella melitensis 16M), NP_639408.1 (Xanthomonas campestris pv. campestris str. ATCC 33913), NP_669902.1 (Yersinia pestis KIM), NP_828458.1 (Strep- tomyces avermitilis MA-4680), CAD61030.1 (Arthrobacter ilicis), and ZP_00227861.1 (Kineococcus radiotolerans SRS30216), while many other proteins, including NP_405150.1 (Yersinia pestis CO92), NP_437232.1 (Sinorhizobium meliloti), NP_533476.1 (Agrobacterium tumefaciens str. C58), NP_744975.1 (Pseudomonas putida KT2440), ZP_00213853.1 (Burkholderia cepacia R18194), ZP_00281771.1 (Burkholderia fungorum LB400), AAM39023.1 (Xanthomonas axonopodis pv. citri str. 306), and YP_070105 (Yersinia pseudotuberculosis) were anno- tated with an N-terminus equivalent to that of human rTSβ. Therefore, we determined whether an equivalent extended N-terminus could be predicted in the human rTS gene. Previously, all available human rTS genomic sequences appeared to contain a sequence gap immedi- ately upstream of the start codon of rTSβ, and the pub- lished 5'-end of the rTS mRNAs was originally determined by RACE (Rapid Amplification of cDNA Ends) analysis of cloned sequences [2]. Thus, a longer N-terminal was not predicted initially, and not expected based upon the exist- ing experimental evidence. However, at the time we started this analysis, one BAC clone, RP11-778P8 [AC021474.1], was found to contain the sequence cover- ing an extended exon region as well as the rest of the gene, although its sequence was in a status of unordered frag- ments. In addition, a GenBank entry for the human rTS gene by Dolnick and Su [AF305057] contains the com- plete 5'-end upstream sequence. Analysis of this sequence by GenScan [7] predicts a start codon upstream of the start codon for rTSβ, yielding an extended N-terminal that is 27 amino acids longer than rTSβ (Fig. 1A). A sequence com- parison between the extended human rTS protein region and the bacterial rTS proteins possessing a longer N-termi- nus revealed a high level of sequence similarity in the extended region. Therefore, we reasoned that a longer N- terminus may exist in all rTS orthologous genes, and this was confirmed by the presence of a possible equivalent extension of the N-terminus in all available rTS genomic sequences (Fig. 1A–E). We named this potential new iso- form rTSγ to distinguish it from rTSβ and rTSα. During our preparation of this manuscript, NCBI released a new Ref- Gene entry for human rTSβ [NM_202758 and NP_059982.1]. This entry, dated Dec-20-2004, predicted a different N-terminus and was subsequently replaced by another entry [NM_017512 and NP_059982.2] on 02- March-2005, which has a deduced protein product identi- cal to that described in this report. While all available ani- mal rTS genes, including those from human, three fish species (fresh and salt water pufferfish and zebrafish), the basal chordate, Ciona intestinalis, and the invertebrate Anopheles gambiae, share the same intron/exon boundary position at least for the first exon/intron junction (Fig. 1A&B), the fungal rTS genes seem to vary in gene structure making them different from the animal genes, as well as from each other (Fig. 1C&D). In addition to the genomic sequences, EST sequences for rTS genes containing sufficient 5'-end sequences were also identified for a few more animal species, including cow, rat, frog, multiple bony fish species, sea squirt, beetles and mites. Multiple sequence alignment analysis revealed that the extended N-termini of rTS genes are conserved from bacteria to human (Fig. 2). Therefore, we believe that the extended N-terminal region represents an ancestral form of the rTS gene products. The extended N-terminus contributes a mitochondrial signal in human rTSγ protein During our search for potential new motifs and/or func- tions contributed by the extended N-terminal region of rTS, we found that this extended sequence was predicted Page 2 of 10 (page number not for citation purposes)