Chang L. distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells. genes in zebrafish. We purified the recombinant proteins and demonstrated that both dreCmas enzymes assemble Pimecrolimus into tetramers and are enzymatically active as well as in cellular systems. Remarkably, however, the dreCmas enzymes showed significant differences not only in terms of substrate specificity but also with respect to their subcellular localization and spatial expression. Although dreCmas1 was transported to the nuclear compartment by a bipartite nuclear localization signal, dreCmas2, in contrast to all other vertebrate CMAS analyzed thus far, remained in the cytoplasm. EXPERIMENTAL PROCEDURES DNA and Protein Sequence Analysis Two homologues were identified in the zebrafish genome using known vertebrate sequences in BLAST searches. Sequences of homologues of other species (supplemental Table 1) were obtained from ENSEMBL and GenBankTM databases or by using BLASTP or TBLASTN algorithms applied to protein, mRNA, and EST databases. In the latter case, overlapping ESTs were downloaded, aligned, and sorted for each species according to sequence identities. Regions of unsure sequencing were deleted, and consensus sequences were inferred manually. For phylogenetic analyses, nucleotide sequences were aligned using CLUSTALW implemented in MEGA (16). The maximum likelihood tree was constructed using Mega5 and tested by bootstrap analysis with 1000 replications. The ambiguously aligned N-terminal and C-terminal codons were excluded from analysis, resulting in a 1221-bp-long (407 amino acids) alignment. All sites in triplets were used, and missing data and alignment gaps were deleted in pairwise comparisons. We used the general time-reversible model of substitutions and uniform rates of substitutions among sites. The tree was inferred using the nearest neighbor interchange maximum likelihood heuristic method. The programs EBI-ClustalW (17) and Bio Edit 7.0.5 (Tom Hall, Ibis Therapeutics, Carlsbad, CA) were used for multiple sequence alignments for visualization of conserved domains and nuclear localization signals. Prediction of putative nuclear localization signal (NLS) sequences was performed by eye and by use of PsortII and PredictNLS. Structure prediction was done using Phyre (18). Blocks of synteny were determined with the help of the Synteny database (19) or on sight using the latest versions of genome projects provided by ENSEMBL database. In the latter case, chromosomal location and orientation of orthologues of up to 10 genes upstream and downstream of the genes of zebrafish were searched for in other species. Only the relative chromosomal position was taken into consideration. We included species abbreviations in the gene names (dreCmas). For hemichordates and fish, we used the nomenclature recommended for zebrafish (gene, and drewere amplified using specific primers (supplemental Table 2) and Phusion DNA Polymerase (Finnzymes). PCR Pimecrolimus products were subcloned into the pCR-Blunt II-TOPO vector (Invitrogen). Their identities were confirmed by sequencing. Cmas Purification and Size Exclusion Chromatography We subcloned dreand drcDNAs into a modified pET22b-Strep vector allowing the expression of N-terminally Strep II-tagged (IBA) proteins. Primer sequences are provided in supplemental Table 2. Recombinant dreCmas1 and dreCmas2 were expressed in BL21(DE3) (Novagen) at 15 C in Power Broth (AthenaES) and purified by Strep-Tactin affinity chromatography (IBA). Peak fractions were desalted (HiPrep 26/10, GE Healthcare) and concentrated to 1C2 mg ml?1 in buffer containing 50 mm Tris-HCl (pH 8), 20 mm MgCl2, 150 mm NaCl, and 1 mm DTT. Purified protein samples were flash-frozen in liquid nitrogen and stored at ?80 C until required. Protein concentrations were determined using the absorption at 280 nm and the Pimecrolimus specific extinction coefficient calculated using the ProtParam tool. Size exclusion chromatography was performed as described previously using the abovementioned buffer (20). Following purification and size ART1 exclusion chromatography, Western blots were stained with Strep-Tactin-alkaline phosphate (AP) conjugate (IBA). In Vitro Activity Assay Sia activation was analyzed.