Danielson K G, Pillarisetti J, Cohen I R, Sholehvar B, Huebner K, Ng L J, Nicholls J M, Cheah K S, Iozzo R V
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1995 Dec 29;270(52):31225-34. doi: 10.1074/jbc.270.52.31225.
We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3'-untranslated region of approximately 6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts. We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning. The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs. It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes. In support of this, primer extension revealed extension two transcription start sites. Transient cell transfection assays showed functional promoter activity for the 3.9-kilobase pair 5'-flanking region. Interestingly, internal and 5' deletions revealed tha the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation. In situ hybridization analyses of early human embryos at 28-42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.
我们报告了人类WNT - 5A基因的完整基因组结构，该基因编码一种富含半胱氨酸的生长因子，参与生长和分化过程中的细胞间信号传导。该基因由五个外显子组成，末端外显子编码一个约6.5千碱基对的大3'非翻译区，并利用多个聚腺苷酸化信号产生至少四种不同的转录本。我们发现了一个新的前导外显子，它被一个411碱基对的内含子打断，该内含子保留在我们最初的cDNA克隆中。启动子区域位于一个富含GpC的岛中，含有许多顺式作用元件，包括几个GC盒以及Sp1、AP1和AP2结合基序。它缺乏管家基因和生长因子基因典型的TATA或CAAT盒。与此相符的是，引物延伸显示有两个转录起始位点。瞬时细胞转染试验表明，3.9千碱基对的5'侧翼区域具有功能性启动子活性。有趣的是，内部和5'缺失显示，完整转录活性并不需要远端启动子，且WNT - 5A的前631个碱基对具有最强的启动子活性。利用一组携带3号染色体短臂部分的啮齿动物 - 人类杂交DNA，我们将该基因定位到3p14.2 - p21.1，位于一个先天性和一个家族性肾细胞癌相关易位之间。对妊娠28 - 42天的早期人类胚胎进行原位杂交分析发现，WNT - 5A转录本不仅局限于发育中的脑和肢体，还在咽裂和咽囊周围的间充质以及发育中的性腺和肾脏中观察到。在体腔上皮以及卵泡和生精小管前体中的相对高表达表明WNT - 5A在生殖细胞分化中具有新的作用。这项研究为识别WNT - 5A基因的调控提供了分子基础，并为研究与这个重要基因相关的遗传疾病提供了机会。
