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Molecular cloning and structural analysis of the functional mouse genomic XPG gene
D L Ludwig1, J S Mudgett, M S Park
1Life Sciences Division, MS M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Abstract:
The mouse XPG gene is a homolog of the human DNA excision repair gene known to be defective in the hereditary sun-sensitive disorder xeroderma pigmentosum (group-G). Defects in mouse XPG have been shown to directly affect the sensitivity of cultured cells to chemotherapy agents and may play a role in tumor cell drug resistance in vivo. A full-length cosmid clone of mouse XPG was isolated by complementation of the UV sensitivity and repair defect in CHO-UV135 cells. Exon mapping determined that the gene consisted of 15 exons within 32 kb of genomic DNA. Sequencing of intron-exon boundaries revealed that mouse XPG possesses a rare class of intron previously identified in only four other eukaryotic genes; it utilizes AT and AC dinucleotides instead of the expected GT and AG within the splice junctions. Promoter analysis determined that mouse XPG is expressed constitutively and probably initiates transcription from multiple start sites, yet, unlike the yeast homolog RAD2, we found no evidence that it is UVC inducible in cultured cells. Amino acid comparison with human XPG identified a highly conserved acidic region of homology not previously described.
Insights
Researchers characterized the mouse XPG gene, a DNA repair gene crucial for preventing sun sensitivity and potentially impacting cancer drug resistance. They identified unique genetic features and constitutive expression, differing from yeast homologs.
Area of Science:
- Genetics
- Molecular Biology
- DNA Repair
Background:
- The mouse XPG gene is homologous to the human gene involved in DNA excision repair, defective in xeroderma pigmentosum.
- Defects in mouse XPG influence cellular sensitivity to chemotherapy and may contribute to in vivo tumor drug resistance.
Purpose of the Study:
- To isolate and characterize the full-length mouse XPG gene.
- To investigate its genomic structure, expression patterns, and homology with human XPG.
Main Methods:
- Complementation of UV sensitivity and repair defects in CHO-UV135 cells using a cosmid clone.
- Exon mapping to determine gene structure.
- Sequencing of intron-exon boundaries.
- Promoter analysis to assess expression and inducibility.
- Amino acid comparison with human XPG.
Main Results:
- A full-length mouse XPG cosmid clone was isolated.
- The gene comprises 15 exons spanning 32 kb of genomic DNA.
- Mouse XPG utilizes rare AT/AC dinucleotides at splice junctions.
- Constitutive expression from multiple start sites was observed, with no UVC inducibility.
- A highly conserved acidic region of homology between mouse and human XPG was identified.
Conclusions:
- The mouse XPG gene has a unique genomic structure and splicing mechanism.
- Its constitutive expression pattern differs from yeast RAD2.
- The identified conserved region may be functionally significant for XPG protein.