Genetic linkage analysis of X-ray hypersensitivity in the LEC
Center for Experimental Animal Science, Nagoya City University Medical School, Nagoya, Aichi 468-8601, Japan
KAC Co., LTD., Kyoto, Kyoto 604-8423, Japan
Department of the First Surgery, Nagoya City University Medical School, Nagoya, Aichi 468-8601, Japan
Received: 14 February 2000 / Accepted: 17 May 2000
Abstract. The LEC rat has been reported to exhibit X-ray hyper-
sensitivity and deficiency in DNA double-strand break (DSB) re-
pair. The present study was performed to map the locus respon-
sible for this phenotype, the xhs (X-ray hypersensitivity), as the
first step in identifying the responsible gene. Analysis of the prog-
eny of (BN × LEC)F
× LEC backcrosses indicated that the X-ray
hypersensitive phenotype was controlled by multiple genetic loci
in contrast to the results reported previously. Quantitative trait loci
(QTL) linkage analysis revealed two responsible loci located on
Chromosomes (Chr) 4 and 1. QTL on Chr 4 exhibited very strong
linkage to the X-ray hypersensitive phenotype, while QTL on Chr
1 showed weak linkage. The Rad52 locus, mutation of which
results in hypersensitivity to ionizing radiation and impairment of
DNA DSB repair in yeast, was reported to be located on the
synteneic regions of mouse Chr 6 and human Chr 12. However,
mapping of the rat Rad52 locus indicated that it was located 23 cM
distal to the QTL on Chr 4. Furthermore, none of the radio-
sensitivity-related loci mapped previously in the rat chromosome
were identical to the QTL on Chrs 4 and 1 in the LEC rat. Thus,
it seems that X-ray hypersensitivity in the LEC rat is caused by
mutation(s) in as-yet-undefined genes.
Ionizing radiation induces DSBs in DNA. Breaks of DNA are
repaired by a Rad52- or Ku-initiated system. In yeast, the former
system contributes mainly to DSB repair, whereas vertebrates use
the latter. The SCID mouse is a well-known animal model of
impaired DNA repair (Fulop and Phillips 1990; Biedermann et al.
1991). SCID mice have been shown to be radio-sensitive owing to
a point mutation in the gene encoding the DNA-dependent protein
kinase catalytic subunit (DNA-PKcs), with which Ku interacts as
a subunit (Blunt et al. 1996; Danska et al. 1996; Araki et al. 1997).
Mice with targeted disruption of genes belonging to the Ku epis-
tasis group involved in DSB repair, such as Ku70, Ku80, DNA-
PKcs, and ligase IV, all show radio-sensitive phenotypes (Gu et al.
1997; Nussenzweig et al. 1996; Zhu et al. 1996; Gao et al. 1998;
Frank et al. 1998).
The LEC rat was established from a closed colony of Long-
Evans rats as a mutant exhibiting fulminant hepatic disorder
(Sasaki et al. 1985; Yoshida et al. 1987). LEC rats were shown to
be defective in differentiation of T cells in the thymus (Agui et al.
1990). Besides these interesting mutations, LEC rats have been
reported to be sensitive to both ionizing radiation and DNA-
damaging agents (Hayashi et al. 1994; Okui et al. 1996). The
radio-sensitive phenotype was shown to be controlled by an auto-
somal single recessive locus, xhs. Further, the repair of DNA DSBs
was reported to be impaired in fibroblasts from LEC rats (Hayashi
et al. 1994). These results suggested that the LEC rat would be a
useful animal model for the study of DNA DSB repair mecha-
nisms. In this study, we performed genetic linkage analysis of the
radio-sensitive phenotype as the first step toward identifying the
gene(s) responsible for the xhs phenotype. We showed in this
paper that the radio-sensitive phenotype in LEC rats is controlled
by multiple genetic loci mapped to Chrs 4 and 1, in contrast to
Materials and methods
LEC/Ncu and BN/Sea rats were maintained in our facility under
specific pathogen-free conditions. The progeny of (BN × LEC)F
× LEC backcrosses were obtained by crossing the above strains.
Animal breeding rooms were maintained at 23 ± 2°C and 50 ± 10% relative
humidity with a 12-hour light-dark cycle (lights on from 8:00 a.m. to 8:00
p.m.). Research was conducted according to the Guideline for the Care and
Use of Laboratory Animals of Nagoya City University Medical School.
The experimental protocol was approved by the Institutional Animal Care
and Use Committee of Nagoya City University Medical School.
All animals were X-irradiated at 5 weeks of age at a dose
rate of 0.2 Gy/min using a Softex M-80WE X-ray generator operating at
100 kVp and 10 mA with a 0.1 mm Cu filter. X-irradiation was performed
between 10:00 a.m. and 2:00 p.m. After X-irradiation, the clinical condi-
tion of all rats was carefully examined twice a day.
Simple sequence length polymorphism analysis.
primers were purchased from Research Genetics, Inc. (Huntsville, Ala.).
Polymerase chain reaction (PCR) was performed with conditions according
to the manufacturer’s instructions. Linkage analysis was performed by
using Map Manager v2.6.5 and Map Manager QTb2168k.
Determination of the thid (T-helper immunodeficiency) genotype.
Blood (50–100 l) was collected from the tail vein of rats and diluted with
an equal volume of phosphate-buffered saline containing 1 UPS std K-II
unit/ml heparin. Blood cells were stained by incubation with fluorescein
isothiocyanate-labeled anti-rat CD4 (OX38, PharMingen, San Diego, Ca-
lif.). After staining, cells were treated with FACS lysing solution (Becton
Dickinson, San Jose, Calif.), and then analyzed with a FACScan cell sorter
(Becton Dickinson, Mountain View, Calif.) with CELLQuest software
(Becton Dickinson, Mountain View, Calif.). The thid genotype was deter-
mined by the ratio of CD4
cells as described previously (Yamada et al.
Detection of polymorphism in the Rad52 gene.
Part of the third
intron of the Rad52 gene was amplified by PCR with primers 5Ј-AGGTC-
AGAAGGTAAGTTGA-3Ј and 5Ј-ATGGAATTTGAAGAGGGCAT-3Ј.
GenBank accession number AF179629.
Correspondence to T. Agui; E-mail: email@example.com.
Mammalian Genome 11, 862–865 (2000).
© Springer-Verlag New York Inc. 2000