An Sp185/333 gene cluster from the purple sea urchin and putative microsatellite-mediated gene diversification.

BACKGROUND: The immune system of the purple sea urchin, Strongylocentrotus purpuratus, is complex and sophisticated. An important component of sea urchin immunity is the Sp185/333 gene family, which is significantly upregulated in immunologically challenged animals. The Sp185/333 genes are less than 2 kb with two exons and are members of a large diverse family composed of greater than 40 genes. The S. purpuratus genome assembly, however, contains only six Sp185/333 genes. This underrepresentation could be due to the difficulties that large gene families present in shotgun assembly, where multiple similar genes can be collapsed into a single consensus gene. RESULTS: To understand the genomic organization of the Sp185/333 gene family, a BAC insert containing Sp185/333 genes was assembled, with careful attention to avoiding artifacts resulting from collapse or artificial duplication/expansion of very similar genes. Twelve candidate BAC assemblies were generated with varying parameters and the optimal assembly was identified by PCR, restriction digests, and subclone sequencing. The validated assembly contained six Sp185/333 genes that were clustered in a 34 kb region at one end of the BAC with five of the six genes tightly clustered within 20 kb. The Sp185/333 genes in this cluster were no more similar to each other than to previously sequenced Sp185/333 genes isolated from three different animals. This was unexpected given their proximity and putative effects of gene homogenization in closely linked, similar genes. All six genes displayed significant similarity including both 5' and 3' flanking regions, which were bounded by microsatellites. Three of the Sp185/333 genes and their flanking regions were tandemly duplicated such that each repeated segment consisted of a gene plus 0.7 kb 5' and 2.4 kb 3' of the gene (4.5 kb total). Both edges of the segmental duplications were bounded by different microsatellites. CONCLUSIONS: The high sequence similarity of the Sp185/333 genes and flanking regions, suggests that the microsatellites may promote genomic instability and are involved with gene duplication and/or gene conversion and the extraordinary sequence diversity of this family.

Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

BACKGROUND: Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. METHODS AND PRINCIPAL FINDINGS: Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3) transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC) lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1) and PC(O-34:1) plasmalogens were displaced by PC(30:0) and PC(32:0) species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. CONCLUSIONS AND SIGNIFICANCE: We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3), expression-specific (Tax1 vs. Tax3) and cell-type-specific (T lymphocytes vs. kidney epithelial cells) changes in the metabolite profiles. The new insight on the affected metabolic pathways can be used to better understand the molecular mechanisms of HTLV induced transformation, which in turn can result in new treatment strategies.