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The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1)
J E Lee1, T W Beck, U Brennscheidt
1Program Resources Inc./Dyncorp., National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Abstract:
The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. We have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776 nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors.
Insights
Researchers identified the ARAF1 gene
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Raf proto-oncogenes encode serine/threonine kinases crucial for cell growth.
- A-raf-1 (ARAF1) is predominantly expressed in urogenital tissues.
- ARAF1 is located on human chromosome Xp11.2, with a pseudogene (ARAF2) on chromosome 7.
Purpose of the Study:
- To isolate and characterize the human ARAF1 gene and its promoter region.
- To understand the transcriptional regulation of ARAF1.
Main Methods:
- Isolation of human genomic DNA clones containing ARAF1 and ARAF2.
- Nucleotide sequence analysis of ARAF1.
- RNase protection and primer extension assays to determine the transcriptional start site.
- Functional assays using CAT reporter gene to confirm promoter activity.
Main Results:
- The ARAF1 gene consists of 16 exons spanning at least 10,776 nucleotides.
- The minimal promoter region (-59 to +93) was identified.
- The ARAF1 promoter lacks TATA and Inr sequences but contains an E box-like element.
- Functional assays confirmed promoter activity.
Conclusions:
- The structural and functional characterization of the ARAF1 gene and its promoter provides insights into its regulation.
- The identified promoter elements may interact with transcription factors like USF and TFII-I.
- This study contributes to understanding the molecular basis of ARAF1's role in cell growth and development.