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Structure and expression of a murine homologue of sky receptor tyrosine kinase gene
Abstract:
To identify new receptor tyrosine kinases (RTKs), we screened cDNAs from mouse mammary tumor cells and mouse brain. A homology search of the complete cDNA sequences obtained showed that one cDNA was a murine homologue of recently reported human sky [Ohashi, K. et al. (1994) Oncogene 9, 699-705]. Another cDNA obtained was also related to sky but had a 5' upstream sequence similar to brt [Fujimoto, J. and Yamamoto, T. (1994) Oncogene 9, 693-698]. Analysis of the 5' region of the sky genomic DNA revealed that brt-type and sky-type sequences are encoded by the sky gene in different exons. The upstream region of the sky-type coding exon is highly GC-rich and contains potential recognition sites for the Sp1 trans-acting factor, but lacks TATA and CAAT boxes, features commonly found in promoters of other RTKs. To examine whether this upstream region functions as a promoter, we fused it with chloramphenicol acetyltransferase (CAT) gene and transfected the construct into COS-7 cells. The results of the CAT assay showed that the sky upstream region retains a significant promoter activity. Furthermore, primer extension analysis revealed that the transcription starts at -240 nt upstream from the sky translation initiation codon. These observations suggest that the brt- and sky-types of mRNA are transcribed from a single sky gene by an alternative promoter usage.
Insights
Researchers identified a new receptor tyrosine kinase (RTK) homologue, sky, in mice. This study reveals alternative promoter usage within the sky gene, leading to different mRNA types.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Receptor tyrosine kinases (RTKs) play crucial roles in cellular signaling and are often implicated in cancer.
- Identifying novel RTKs is essential for understanding complex biological pathways and developing targeted therapies.
Purpose of the Study:
- To identify novel receptor tyrosine kinases (RTKs) in mouse mammary tumor cells and brain.
- To characterize the genetic and regulatory mechanisms underlying the newly identified sky gene and its variants.
Main Methods:
- Screening of cDNA libraries from mouse mammary tumor cells and brain.
- Homology searches and sequence analysis of obtained cDNA.
- Genomic DNA analysis of the 5' region of the sky gene.
- Reporter gene assays (chloramphenicol acetyltransferase - CAT assay) to assess promoter activity.
- Primer extension analysis to determine transcription start sites.
Main Results:
- Identification of a murine homologue of the human sky RTK.
- Discovery that sky and brt-type sequences originate from the same sky gene, encoded by different exons.
- The upstream region of the sky-type coding exon exhibits promoter activity, independent of TATA/CAAT boxes.
- Transcription initiation site identified at -240 nt upstream of the sky translation start codon.
Conclusions:
- The sky gene utilizes alternative promoter usage to generate distinct mRNA variants (sky-type and brt-type).
- This alternative promoter mechanism provides a novel regulatory strategy for RTK expression.
- Findings contribute to a deeper understanding of RTK gene regulation and potential therapeutic targets.