Mapping a novel cellular-senescence gene to human chromosome 2q37 by irradiation microcell-mediated chromosome transfer.
To identify the subchromosomal region that carries the cellular-senescence-restoring program of the human cervical carcinoma cell line SiHa, we constructed by irradiation microcell-mediated chromosome transfer a library of mouse A9 cells containing various fragments of human chromosome 2 tagged with pSV2neo in 2p11-p12. Eighty-seven clones were isolated and screened for the presence of human sequences by inter-Alu and inter-L1 polymerase chain reaction (PCR), and six clones exhibiting PCR-laddering patterns that differed from those of the A9 cells containing an intact chromosome 2 were examined further. Chromosome analysis and fluorescence in situ hybridization (FISH) using human-specific repetitive sequences revealed that four of these clones contained single subchromosomal transferable fragments (STFs). Southern blot hybridization of 14 cosmid markers revealed that the STFs in A9 cells were derived from human chromosome 2. These STFs were transferred into SiHa cells by microcell fusion, and one of the STFs restored the cellular-senescence program. The concordance of the cellular-senescence-restoring program with the presence or absence of specific DNA fragments of chromosome 2 indicated that the putative cellular-senescence gene was located in 2q32-qter. For more detailed mapping, we constructed mouse A9 cells containing STFs derived from human chromosome 2 tagged with pSTneo at different regions in 2q31-qter. PCR-laddering and FISH analyses were used to identify six clones that contained different STFs. These STFs were transferred into SiHa cells, and one of the three clones that restored cellular senescence contained a small fragment of human chromosome 2. This STF was shown by PCR analysis using 14 human chromosome 2-specific primer pairs to be smaller than 12.2 cM and was mapped to the 2q37 region by FISH analysis with inter-Alu PCR. Beta-galactosidase activity, which is a biomarker of senescent cells, and telomerase activity similar to that found in parental SiHa cells were detected in SiHa microcell hybrids, suggesting that the putative cellular-senescence gene was not involved in a telomerase pathway but rather in an alternate pathway of cellular senescence.
Uejima
H
H
Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, and CREST (JST), Yonago, Japan.
Shinohara
T
T
Nakayama
Y
Y
Kugoh
H
H
Oshimura
M
M
D000818:Animals; D002460:Cell Line; D016922:Cellular Senescence / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D002889:Chromosomes, Human, Pair 2; D003360:Cosmids; D017931:DNA Primers; D005260:Female; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007621:Karyotyping; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D016324:Sequence Tagged Sites; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms
D017931:DNA Primers; D005456:Fluorescent Dyes; D005819:Genetic Markers