Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U39837
This view has gained support from a number of experimental observations, including the fact that some proto-oncogenes code for growth factors, that mutant forms can arrest, distort, or promote differentiation, and that they are temporally and spatially sequestered during embryogenesis [9]
We investigated the genomic organization, promoter activity, and chromosomal localization of the WNT-5A gene, which codes for a cysteine-rich growth factor involved in cell-cell signaling during embryonic development and perhaps tumor growth
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U39837. To facilitate the study of human WNT-5A gene and to understand its transcriptional control, we have characterized the entire genomic organization of WNT-5A, established functional promoter activity for the 5Ј-flanking region in transient cell transfection assays, determined a finer chromosomal location for the gene, and investigated its expression in early human embryogenesis. 1. Heterogeneity of the 5 end of the WNT-5A cDNA (A) and determination of the transcription start sites in the WNT-5A gene by primer extension (B) and RT-PCR (C).
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