The interspecific backcross established by Kay et al
(1991) was utilised in order to determine the position of the calbindin D9K gene on the mouse X chromosome with respect to the hyp locus. The backcross was established using 2 evolutionary divergent mouse species. Mus musculus domesticus
and Mus spretus, and in order to investigate recombination events and determine the molecular map position for a marker, a restriction fragment length variant or RFLV between the two mouse species for the calbindin cDNA probe was sought.
Mus musculus domesticus and Mus spretus mouse genomic DNA was digested with the following enzymes: TaqI, MspI, PstI, PvuII, EcoRI, EcoRV, Hindlll, Bglll and BamHI. The digested DNA fragments were resolved by agarose gel electrophoresis and transferred to a nylon membrane by Southern blotting. The "polyblot" was then hybridised with the rat calbindin cDNA probe, and autoradiographed. Analysis of the results obtained revealed an RFLV between Mus musculus domesticus and Mus spretus with the enzyme TaqI, with allele sizes of 2.2 and 1.9 Kilobases respectively (Figure 6.2).
Genomic DNA for male and female progeny from the interspecific backcross was digested with TaqI and Southern blots of the digested DNA prepared. The blots were hybridised with the rat calbindin cDNA, as described in Materials and Methods, and autoradiographed. An example of the results obtained for this analysis of the male progeny from the interspecific backcross is shown in Figure 6.2.
Mouse Backcross Number
22 24 35 39 40 41 51 52 58 60 80 88 89 90 100 D S
Size
(Kb)
2.2
—1.9 -
Figure 6.2: Autoradiograph of Southern blot of TaqI digested male backcross progeny DNA hybridised with rat calbindin cDNA to reveal Mus musculus domesticus and Mus spretus RFLV alleles. The mouse backcross number is shown above the revealed domesticus (size 2.2 Kb) or spretus (1.9 Kb) allele. D: Control Mus musculus domesticus DNA; S: Control Mus spretus DNA. The presence of a domesticus
or spretus allele for each animal from the backcross panel was therefore ascertained for
segregation analysis and localisation of the calbindin gene on the mouse X chromosome with respect to the hyp locus.
The results obtained for the scoring of the key male and female backcross progeny are shown in Figures 6.3 and 6.4 respectively. For the male mice, there are recombination events between calbindin and the pgk-1 and DXSmh43 loci thereby indicating a distal location of calbindin to these two markers. In addition, recombination is observed between the Amg locus and calbindin, demonstrating that calbindin is proximal with respect to the Amg l o c u s . For calbindin and the marker Cbx-rsl, there are no recombination events between these two markers and thus they cannot be ordered. The localisation of the calbindin gene with respect to the hyp
locus, however, demonstrates that two mice (backcross numbers 22 and 24) are recombinant between the two markers.
Analysis of the female progeny (Figure 6.4) confirms the localisation of the calbindin gene distal to DXSmh43 and proximal to Amg. A recombination event between calbindin and Cbx-rsl (mouse backcross number 187) allows the localisation of calbindin proximal to Cbx-rsl.
Thus, the combined male and female results of the mapping of the calbindin gene using the interspecific backcross segregating for hyp indicate the locus order:
Backcross Mouse Number Locus 183 240 41 164 184 22 24 35 51 58 89 100 118 223 238 105 90 120
■ ■ ■ ■ ■ ■ ■ □ □ □ □ □ □ □ □ □ □ □
D X S m h 4 3 □ □ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ □ □ □□ □ □ □ - ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ □ □
CALB.ND1N
□ □ □ □ □ □ □ b b b h h h b
B B D D
□ □ □ □ □ □ □ ■ ■ ■ ■ ■ ■ ■ ■ ■ □ □
Cbx-rsl ''"’9□ □ □ □ □ □ □
□
Soretus alleleProposed locus order: pgk1 - Dxsmh43 - hyp - (Calbindin, Cbx-rsl) - Amg. Domesticus allele
Spretus allele Genotype unknown
Figure 6.3: Molecular mapping of the calbindin D9K gene on the mouse X chromosome in male progeny of the interspecific backcross segregating for hyp. The results obtained for haplotype analysis with the calbindin cDNA probe for key male mice, denoted as their backcross mouse number, together with the loci pgk-1, DXSmh43, hyp, Cbx-rsl and Amg (Kay et al 1991) are shown. Analysis of recombination events allows the localisation of calbindin distal to DXSmh43 and proximal to Amg. There are no recombination events between calbindin and Cbx-rsl, and thus the order of the two markers cannot be ascertained. Localisation of the calbindin gene with respect to hyp demonstrates two recombinants (backcross mouse numbers 22 and 24) .
Backcross Mouse Number Locus 171 113 78 213 219 4 46 82 84 96 101 125 126 156 161 210 77 86 187 188 1 6
pgk-1
■ ■ ■ ■ ■ □ □ □ □ □ □ □ □ □ □ □ □ □ □ □ □ □
D X S m h 4 3 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ □ □ □ □ □ □ hyp ... CALBINDIN □ □ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ □ ■ □ □I I ’ H
i
l
l
■ l
l
l
l
l
l
l
l
l
l
l
l
l
l
I I I
A m gOOOOOIIIIIIIIIIIIIIIII
I I Domesticus alleleI
Spretus alleleProposed locus order: pgk1 - D X S m h 4 3 - hyp - Calbindin - Cbx-rsl - Amg. Genotype unknown
Figure 6.4: Molecular mapping of the calbindin D9K gene on the mouse X chromosome in female progeny of the interspecific backcross. The results obtained for haplotype analysis using the calbindin cDNA probe for key female mice, denoted as their backcross mouse number, together with the loci pgk-1, DXSmh43, Cbx-rsl and Amg (Kay et al 1991) are shown. Analysis of recombination events confirms the localisation of calbindin distal to DXSmh43 and proximal to Amg. Recombination between calbindin and Cbx-rsl (backcross mouse number 187) allows the order of the two loci to be ascertained, with calbindin mapping proximal to Cbx-rsl. The hyp phenotype could not be ascertained unequivocally in female mice, and this is therefore shown as blank (-).
The use of the interspecific backcross, mapping the calbindin D9K gene proximal to the Cbx-rsl locus, demonstrates that calbindin is the closest identified distal marker to the
hyp locus on the mouse X chromosome.
The finding of recombination between the calbindin gene and the hyp locus does not necessarily exclude an association between the two loci. Although the biochemical analysis of control domesticus mice demonstrated (Eicher at al 1976, Kay
at al 1991) that the hyp mutation resulted in significant hypophosphataemia (and raised alkaline phosphatase activity) in both males and females, it may still be possible for diagnostic confusion in the progeny from the backcross. Thus the calbindin D9K gene may represent a candidate locus for the
hyp mutation and the sequence of the calbindin D9K gene in the
hyp mouse therefore investigated.