A gene trap approach to isolate mammalian genes involved in the cellular response to genotoxic stress.
Treatment of cells with DNA damaging agents leads to induction of a variety of genes involved in different cellular processes. We have applied a lacZ-based gene trap strategy to search for new mammalian genes induced by genotoxic stress. A population of 32 x 10(3) neo r clones stably transfected with a gene trap vector was obtained, stained with fluorescein di-beta-d-galactopyranoside and analyzed by flow activated cell sorting and replica plating. This strategy allowed isolation of 30 neo r 'putative inducible' cell lines expressing lacZ only after a DNA damaging treatment. For three clones the site of integration and the degree of inducibility after UV treatment were determined by Southern blot and beta-galactosidase measurement respectively. One cell line (clone VI) showed a single integration site and a reproducible 3-fold induction of beta-galactosidase activity following UV irradiation. Fused transcripts were isolated from induced cells and a portion of the trapped gene was amplified by rapid amplification of cDNA ends. Sequence analysis and comparison with available gene and protein databanks revealed that the gene was novel.
Menichini
P
P
CSTA-Laboratory of Mutagenesis, National Institute for Research on Cancer (IST), Largo Rosanna Benzi, 10, Genoa, Italy. menikini@hp380.ist.unige.it
Viaggi
S
S
Gallerani
E
E
Fronza
G
G
Ottaggio
L
L
Comes
A
A
Ellwart
J W
JW
Abbondandolo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004249:DNA Damage; D005821:Genetic Techniques; D005822:Genetic Vectors; D017404:In Situ Hybridization, Fluorescence; D007763:Lac Operon; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004247:DNA; D001616:beta-Galactosidase