cDNA Cloning of a Novel WD Repeat Protein Mapping to the 9q22.3 Chromosomal Region

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Abstract
To identify expressed sequences from the candidate genomic region of the nevoid basal cell carcinoma syndrome (9q22.3), the CpG island rescue PCR methodology was employed on the yeast artificial chromosome (YAC) ICI-8AD8 that contains microsatellite marker D9S180. A positive clone (IR10, size 350 bp) was isolated by screening a human epidermal cDNA library with the island rescue PCR products and mapped back to the 9q22.3 region. To obtain additional sequence information from IR10, the rapid amplification of cDNA ends (RACE) methodology was employed. Within the longest IR10 RACE product, two segments having similarities with coronin, a G-like and actin-binding protein of Dictyostelium discoideum, as well as with p57, a recently cloned human actin-binding protein, were revealed. In fact, these two segments have properties of typical exons, suggesting that the RACE products and clone IR10 represent unspliced pre-mRNAs. Using the RACE methodology with primers originating from these two exons, it was shown that they splice together and the sequences corresponding to the processed IR10 mRNA were revealed. This mRNA maps back to 9q22.3 and furthermore was found to code for a novel protein composed of 525 amino acids and containing five WD repeats. The presence of the WD repeat motif, considered to function in protein–protein interactions, implies that the new protein might have a role in intracellular signaling. Moreover, Northern analysis indicated that, in addition to epidermis, the brain is a tissue with relatively high levels of expression of this gene. Finally, Southern analysis showed a functional conservation of the WD repeat protein in various species.