A high-resolution radiation hybrid map of the proximal region of rat
Khulood M. Al-Majali,
* Anne M. Glazier,
* Penny J. Norsworthy,
Faisal N. Wahid,
Lisa D. Cooper,
Caroline A. Wallace,
Timothy J. Aitman
Molecular Medicine Group, MRC Clinical Sciences Centre, Hammersmith Hospital, DuCane Road, London W12 ONN, UK
Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK
Division of National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK
Department of Medical Statistics and Evaluation, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK
Received: 4 December 1998 / Accepted: 19 January 1999
Abstract. Radiation hybrid (RH) mapping has been used to pro-
duce genome maps in the human and mouse, but as yet the tech-
nique has been applied little to other species. We describe the use
of RH mapping in the rat, using a newly available rat/hamster RH
panel, to construct an RH map of the proximal part of rat Chro-
mosome (Chr) 4. This region is of interest because quantitative
trait loci (QTLs) for defective insulin and catecholamine action,
hypertension, and dyslipidemia map to this region. The RH map
includes 23 rat genes or microsatellites previously mapped to this
part of Chr 4, one rat gene not previously mapped in the rat, and
markers for four new genes, homologs of which map to the syn-
tenic region of the mouse genome. The RH map integrates genetic
markers previously mapped on several rat crosses, increases the
resolution of existing maps, and may provide a suitable basis for
physical map construction and gene identification in this chromo-
somal region. Our results demonstrate the utility of RH mapping in
the rat genome and show that RH mapping can be used to localize,
in the rat genome, the homologs of genes from other species such
as the mouse. This will facilitate identification of candidate genes
underlying QTLs on this chromosomal segment.
Radiation hybrid (RH) mapping is a somatic cell hybrid technique
that was developed to construct high-resolution, contiguous maps
of mammalian chromosomes. The technique provides a method for
ordering DNA markers spanning millions of base pairs of DNA at
a resolution not easily obtained by other mapping methods (Cox et
al. 1990; Burmeister et al. 1991; Warrington et al. 1992; Abel et al.
1993). An advantage of radiation hybrid mapping is the ability to
map non-polymorphic DNA markers that cannot be used for mei-
In this method, a lethal dose of X-irradiation is used to break
the chromosomes of the donor cell line into numerous fragments.
Chromosome fragments from the donor cell line are subsequently
retained non-selectively after cell fusion with a recipient cell line.
The resulting hybrid clones are then tested for the retention or loss
of specific donor chromosome markers. Markers that are further
apart on a chromosome are more likely to be broken apart by
radiation and to segregate independently in the RH cells than
markers that are closer together. By analyzing the cosegregation of
various loci in hybrid clones statistically, a map can be constructed
giving information about the relative order and distance of markers
(Cox et al. 1990; Warrington et al. 1991; Ceccherini et al. 1992).
The rat provides one of the most powerful experimental sys-
tems for the study of physiological and pathophysiological traits.
Quantitative trait loci (QTLs) for several such traits have recently
been identified in the rat by genetic analysis (Hilbert et al. 1991;
Jacob et al. 1991; Deng and Rapp 1992; Cicila et al. 1993; Brown
et al. 1996; Galli et al. 1996; Gauguier et al. 1996; Aitman et al.
1997). Rat Chr 4 is of significant interest because QTLs for de-
fective insulin action, catecholamine action, hypertension, and
dyslipidemia have been identified in the proximal region of this
chromosome in the spontaneously hypertensive rat (SHR) (Prave-
nec et al. 1995; Schork et al. 1995; Bottger et al. 1996; Aitman et
al. 1997). These SHR traits are also key features of human Meta-
bolic Syndrome X, for which SHR has been proposed as a model
(Aitman et al. 1997).
In this report, we describe the use of radiation hybrid mapping
in the rat, using a newly available rat/hamster RH panel, to con-
struct an RH map of the proximal part of rat Chr 4. This map
integrates the existing genetic maps derived from several rat
crosses, resolving ambiguities in distance and location of markers
on these maps. The map may provide a suitable basis for construc-
tion of a physical map of this region. Finally we have exploited the
close synteny relationship between mouse and rat to show that
markers for mouse genes can be rapidly and accurately mapped
onto the rat genome.
Materials and methods
Radiation hybrid panel.
The whole genome rat/hamster RH panel of
106 hybrid DNAs was purchased from Research Genetics (Huntsville,
Ala.). The panel was constructed by fusing irradiated cells from a Sprague
Dawley fibroblast line (RatFR) with a recipient hamster line (A23). FR
donor cells were irradiated with 3000 rad prior to fusion with A23 recipient
DNA marker analysis.
Twenty-four rat microsatellite markers from
various sources (Goldmuntz et al. 1995; Jacob et al. 1995; Simon et al.
1996; http://www-genome.wi.mit.edu/rat/public/) were used to screen do-
nor and recipient DNA for variability between FR and A23 (Table 1). The
primer sequences for ILG6 were obtained from Research Genetics: ILG6F
5Ј-TGAGTTCCAGGATACCCAGG-3Ј; ILG6R 5Ј-AAGCGGAGT-
CAAAATACTTTGC-3Ј. The primers for the Nos3 microsatellite were
designed from the rat genome sequence for Nos3 (Hubner et al. 1995):
Nos3F, 5Ј-ACGTTCCTCCTCAGCCCTGG-3Ј; Nos3R, 5Ј-GTGCATG-
TCTGCATAAACATG-3Ј. Oligonucleotide primers were synthesized by
Genosys Biotechnology (Cambridge, UK). Oil-free PCR was carried out
on a TouchDown sub-ambient thermal cycler (Hybaid, Teddington, UK)
with an initial denaturation at 94°C for 3 min, followed by 35 cycles of
denaturation at 94°C for 30 s, annealing at the appropriate temperature
(Table 1) for 45 s, and extension at 72°C for 1 min, with a final extension
* These authors contributed equally to this research.
Correspondence to: T.J. Aitman
Mammalian Genome 10, 471–476 (1999).
© Springer-Verlag New York Inc. 1999