Abstract
We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12. 2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.
Highlights
The ionizing radiation-sensitive mammalian cell lines reported to date can be divided into at least six complementation groups [1]
Employing the cell fusion technique, we found that SX9 cells belong to a complementation group different from that of radiosensitive mammalian cell lines irs-1,2,3 and L5178Y-S [28]
The results showed that SX9 cells exhibit a severe defect in signal joint formation as well as in coding joint formation
Summary
Cell Culture—SX9, SX10, and SR-1 cells were derived from mouse mammary carcinoma FM3A cells. SR-1 cells were utilized as a wild-type reaction; RAG, recombination activating gene; RSS, recombination signal sequence; LA-PCR, long and accuracy-PCR; CHO, Chinese hamster ovary. Hybrid cells grown in suspension were plated in 0.3% soft agar medium containing 10% fetal bovine serum and HAT/ouabain. Sequence of the Whole Region of dna-pkcs and Ku70 cDNA—RT-PCR and sequence of dna-pkcs and Ku70 cDNA were performed as described previously [11]. Construction of Full-length Murine dna-pkcs Expression Vector—The full-length cDNA of murine dna-pkcs was amplified from first strand DNA prepared from SR-1 total RNA with primers AatII-(Ϫ4)MuDNAPK38(ϩ) (5Ј-AAGACGTCGGTGATGGCGGAGGAGGGAACCGGCGTACG-3Ј) and SpeI-12438-MuDNAPK37(Ϫ) (5Ј-GACTAGTAGGAGGTCCTCTCGGAGACAGAATGCTTTA-3Ј), using LA-PCR kit version 2 (Takara). DNA sequence was determined with the appropriate primer set
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