BMC Genomics 2005, 6:125 http://www.biomedcentral.com/1471-2164/6/125 sequences for analysis, were retrieved. Extended N-termini were deduced by extending the start codon to the next available "ATG" upstream of the start codon used by the existing annotation. In addition to the rTS orthologous genomic sequences, we also searched the NCBI EST data- base by performing a TBLASTN search with the extended human rTSβ protein sequence to collect rTS cDNA sequences from additional species. To predict any poten- tial new function or cellular location contributed by the extended N-terminus, we analyzed the human rTSγ and rTSβ protein sequences with Mitoprot [22], TargetP [23], PSORTII [24], and Mitopredict [25,26]. Subcellular fractionation of CCRF-CEM cells The human T-lymphoblastic cell line CCRF-CEM was cul- tured as described [27]. Subcellular fractions of CCRF- CEM cells were isolated essentially as described [28], except that protease (0.5 mM Pefabloc) and phosphatase inhibitors (1 mM sodium metavanadate and 1 mM NaF) were included in all buffers. All steps were performed at 0 – 4°C. Briefly, 1-liter of CCRF-CEM cells (~3.5 × 105 cells/ ml) was centrifuged at 1000 × g for 5 min and the pellet was washed with iced 0.9% NaCl, and then suspended in 5 pellet volumes of ice-cold hypotonic buffer [30] and allowed to swell for 5 min on ice. The suspension was homogenized in an ice-cold 7-ml glass Dounce homoge- nizer with 15 strokes of the tight pestle to obtain > 95% cell disruption. The homogenate was immediately made up to 250 mM sucrose and centrifuged at 1000 × g for 5 min. The pellet was washed once with 2.5 original pellet volumes of cold isotonic buffer (1 mM Na2-EDTA, 250 mM sucrose; pH 6.9). The two supernatants were com- bined to generate the post-nuclear supernatant (PNS). The PNS was centrifuged at 17,000 × g, 15 min to generate a cytosolic fraction (supernatant) and an organellar pellet containing both mitochondria and lysosomes. The organellar pellet was suspended in 1 ml of HES (20 mM HEPES-NaOH, 1 mM Na2-EDTA, 250 mM sucrose; pH 7.4). Subcellular fractions were assayed in duplicate for activity of the cytosolic enzyme lactate dehydrogenase (LDH) [30] and mitochondrial matrix enzyme glutamate dehydrogenase (GDH) [31] to ensure that fractionation was successful. To separate mitochondria and lysosomes, this organellar suspension was made up to 30% (w/v) iodixanol [8,32] in a final volume of 2.2 ml and placed on a 5–20% linear iodixanol gradient (9 ml), centrifuged at 70,000 × g for 1.5 hr, and collected as 0.5-ml fractions, which were then diluted with 0.5 ml ice-cold HES and centrifuged at 30,000 × g for 15 min. The resulting organelle pellets were suspended in 200 µ l of isotonic buffer (as above, except that NaF was 50 mM). Protein concentration was determined using the BioRad (Her- cules, CA) protein assay kit. Western blotting Proteins were resolved by denaturing gel electrophoresis using 10% polyacrylamide and transferred to PVDF mem- branes essentially as described [4]. The primary antibodies used were: D3 (mouse monoclonal to rTSβ), LAMP-1 (mouse monoclonal to lysosome-associated membrane protein-1; Santa Cruz Biotechnology), and MnSOD (rab- bit polyclonal to manganese superoxide dismutase; Stress- gen). Secondary antibodies consisted of horseradish peroxidase conjugated F(ab')2 fragments and were obtained from Jackson ImmunoResearch Laboratories. Probed blots were imaged using West Pico Dura chemilu- minescent reagent (Pierce) and X-OMAT AR X-ray film (Kodak). Pre-stained protein molecular weight markers (BioRad) were included during electrophoresis to allow determination of apparent molecular weights of detected antigens. All experiments were repeated at least twice with similar results. Authors' contributions PL conceived the project, performed most of the compu- tational analysis, and drafted the complete manuscript; BJD conceived the project and carried out the western blot experiments; JRN and JJM performed the subcellular frac- tionation and related enzyme assays; LS contributed to the collection and annotations of rTS sequences. Additional material Additional File 1 Deduced rTS protein sequences based on existing DNA sequences and modifications made to existing rTS protein sequences. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-6-125-S1.doc] Acknowledgements The authors would like to thank Dr. John M. Graham, JG Research Consul- tancy, UK, for his suggestions and advice for the preparation of iodixanol gradients. This work is partially supported by a developmental fund from Roswell Park Cancer Institute (PL) and NIH grants CA101515 (PL), EB002116 (BJD), CA43500 (JJM), and CA16056 (RPCI). References 1. Babbitt PC, Hasson MS, Wedekind JE, Palmer DR, Barrett WC, Reed GH, Rayment I, Ringe D, Kenyon GL, Gerlt JA: The enolase super- family: a general strategy for enzyme-catalyzed abstraction of the α-protons of carboxylic acids. Biochemistry 1996, 35:16489-16501. 2. Dolnick BJ: Cloning and characterization of a naturally occur- ring antisense RNA to human thymidylate synthase mRNA. Nucleic Acids Res 1993, 21:1747-1752. 3. Dolnick BJ, Black AR, Winkler PM, Schindler K, Hsueh CT: rTS gene expression is associated with altered cell sensitivity to thymi- dylate synthase inhibitors. Adv Enzyme Regul 1996, 36:165-180. 4. Dolnick BJ, Black AR: Alternate splicing of the rTS gene prod- uct and its overexpression in a 5-fluorouracil-resistant cell line. Cancer Res 1996, 56:3207-3210. Page 9 of 10 (page number not for citation purposes)