Recent studies have shown that the immune system of the sea urchin, Strongylocentrotus purpuratus, is more complex than previously thought. Exemplifying this is the Sp185/333 gene family, which is upregulated in immunologically challenged animals. The Sp185/333 genes are small (<2kb) with two exons and are members of a large diverse family composed of greater than 40 genes. The current S. purpuratus genome (v2.1), 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. Therefore, to understand the genomic spatial organization of the Sp185/333 gene family, a BAC containing Sp185/333 genes was assembled, paying careful attention to artifacts that may result from very similar genes. Twelve candidate BAC assemblies were generated with varying parameters to account for potential gene collapse or artificial duplication/expansion. PCR, restriction digests, and subclone sequencing were used to identify and validate the optimal assembly. The validated assembly contained six Sp185/333 genes ranging in size from 1.3 to 1.8 kb. These genes 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 characterized by the presence of microsatellites. The three genes not associated with the tandem duplication displayed smaller conserved flanking regions ranging from 400 to 600 nt 5’ and 400 nt 3’ of each gene. The high sequence similarity of the Sp185/333 genes and flanking regions, suggests that the microsatellites promote genomic instability and increase the rate of gene duplication of this family, thereby contributing to its extraordinary diversity.
|Advisor:||Smith, Lizbeth Courtney|
|Commitee:||Donaldson, Robert, Smith, Lizbeth C.|
|School:||The George Washington University|
|Department:||Genomics and Bioinformatics|
|School Location:||United States -- District of Columbia|
|Source:||MAI 48/02M, Masters Abstracts International|
|Keywords:||Gene duplication, Microsatellites, Shotgun assembly, Strongylocentrotus purpuratus|
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