Zaphiropoulos P G, Toftgård R
Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum, Huddinge, Sweden.
DNA Cell Biol. 1996 Dec;15(12):1049-56. doi: 10.1089/dna.1996.15.1049.
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.
为了从痣样基底细胞癌综合征（9q22.3）的候选基因组区域中鉴定出表达序列，我们采用了CpG岛拯救PCR方法，对包含微卫星标记D9S180的酵母人工染色体（YAC）ICI - 8AD8进行分析。通过用岛拯救PCR产物筛选人表皮cDNA文库，分离出一个阳性克隆（IR10，大小为350 bp），并将其定位回9q22.3区域。为了从IR10获得更多的序列信息，我们采用了cDNA末端快速扩增（RACE）方法。在最长的IR10 RACE产物中，发现了两个与冠蛋白（一种盘基网柄菌的G样肌动蛋白结合蛋白）以及与p57（一种最近克隆的人肌动蛋白结合蛋白）具有相似性的片段。事实上，这两个片段具有典型外显子的特性，表明RACE产物和克隆IR10代表未剪接的前体mRNA。使用源自这两个外显子的引物进行RACE方法，结果表明它们可以拼接在一起，并揭示了与加工后的IR10 mRNA相对应的序列。该mRNA定位回9q22.3，并且进一步发现它编码一种由525个氨基酸组成且含有五个WD重复序列的新蛋白质。WD重复基序被认为在蛋白质 - 蛋白质相互作用中发挥作用，这意味着这种新蛋白质可能在细胞内信号传导中起作用。此外，Northern分析表明，除了表皮外，大脑是该基因表达水平相对较高的组织。最后，Southern分析显示WD重复蛋白在各种物种中具有功能保守性。
