| First Author | Shellam GR | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 3 | Pages | 572-574 |
| Mgi Jnum | J:14807 | Mgi Id | MGI:62968 |
| Citation | Shellam GR, et al. (1993) Characterisation of allelic forms at the retinal degeneration (rd) and beta-glucuronidase (Gus) loci for the mapping of the flavivirus resistance (Flv) gene on mouse chromosome 5. Mouse Genome 91(3):572-574 |
| abstractText | Full text of Mouse Genome contribution: CHARACTERISATION OF ALLELIC FORMS AT THE RETINAL DEGENERATION (rd) AND Beta-GLUCURONIDASE (Gus) LOCI FOR THE MAPPING OF THE FLAVIVIRUS RESISTANCE (Flv) GENE ON MOUSE CHROMOSOME 5. GR Shellam, N Urosevic, MY Sangster, JP Mansfield and JS Mackenzie. Dept of Microbiology, University of Western Australia, Nedlands 6009, Western Australia. Introduction Genetically determined resistance to flaviviruses in mice is due to the effect of a single, autosomal dominant gene (1, 2) which has been assigned the symbol Flv, with the resistance and susceptibility alleles being designated Flvr and Flvs respectively (3). The flavivirus resistance allele carried by PRI mice (1) was introduced into the susceptible C3H/He strain to produce the congenic, flavivirus-resistant strain C3H.PRI-Flvr (C3H/RV; 4). Although Flv has not been mapped to a particular chromosome, the observation that these congenic C3H strains carry different alleles at the chromosome 5 locus Ric which controls resistance to Rickettsia tsutsugamushi (5, 6) suggests that Flv may be located on chromosome 5 since the differences between C3H/He and C3H/RV at Ric are probably the result of coinheritance of the allele linked to the selected allele Flvr. As a preliminary to comprehensive studies to map the Flv locus we have sought to confirm its chromosome 5 location by identifying other chromosome 5 differences between C3H/HeJ and C3H/RV mice. The retinal degeneration (rd) locus is tightly linked to Ric (5, 7), and the defective rd allele at this locus is responsible for the recessive trait, retinal degeneration, which can be diagnosed histologically (8). We here present evidence, obtained from histological examination and from Southern blot analysis using an rd probe, that C3H/RV mice carry the wild-type allele at the rd locus, presumably inherited from the PRI strain which provided the Flvr allele, while C3H/HeJ have the defective allele. Thus Flv is linked to rd and Ric on chromosome 5. The determination of the position of Flv requires the use of anchor loci on this chromosome, such as Pgm-1, Eta-1 (a locus synonomous with Ric; 9), rd and Gus-s. As a prelude to mapping studies, the allelic forms of the Gus-s gene were characterised in C3H/HeJ, C3H/RV and other mouse strains by a technique we have developed that employs the polymerase chain reaction (PCR) to amplify a polymorphic region in intron 4 of the gene. This PCR-based method is technically simpler than Southern hybridization which has been used in studies of Gus-s alleles in other strains, and provides a rapid method for use in mapping studies. Materials And Methods Mice: Specific pathogen-free inbred C3H/HeJ, C3H/RV, BALB/c, C57BL/6, (C3H/HeJ x C3H/RV)Fl, (BALB/c x C3H/RV)Fl and (C57BL/6J x C3H/RV)F1 mice, were obtained from the Animal Resources Centre, Murdoch, Western Australia. Mice of both sexes from 3 to 6 months of age were used. Histological Examination: For the assessment of retinal degeneration, eyes were fixed in 2.5% gluteraldehyde and embedded in plastic and 2 micron sections were cut and stained with haematoxylin and eosin. The presence or absence of the photoreceptor cell layer in the retina was determined by light microscopy. Ten mice per strain were used in these studies. Southern blot hybridization: Genomic DNA was extracted from the livers, and digested with Msp I (Promega), electrophorised on 0.8% agarose gels, and transferred to nylon membranes (Hybond N+; Amersham), and hybridised with the rd cDNA probe zr.408 (10), kindly provided by Debora Farber, Jules Stein Eye Institute, Los Angeles, California. Hybridization was performed as described (10). The cDNA probe was labelled using a GIGAprime DNA labelling kit (Bresatec) and [alpha-32p]dCTP(3000 Ci/mmol; Amersham). Autoradiographic images of the blots were obtained after 2 to 7 days at Ð75 degrees C using Kodak XAR-5 and Fuji films. Polymerase chain reaction: To differentiate alleles of Gus-s, the polymerase chain reaction (PCR) was used to amplify a polymorphic region in intron 4 of the gene. A 206-bp insert in this region of the Gus-sa allele in BALB/c mice (11) is absent from other alleles (12). PCR primers of 20 nucleotides were selected from Beta-glucuronidase structural gene sequences flanking the insert (11). The 5' and 3' primers, starting at nucleotides 3165 and 3445, respectively, were synthesised and purified using a 380 B DNA synthesiser and oligonucleotide purification cartridge (Applied Biosystems). Amplification cycles of PCR, performed in a DNA thermal cycler (Perkin-Elmer, Cetus), consisted of an initial denaturing step at 94 degrees C for 5 min., followed by 30 cycles, each consisting of 94 degrees C for 1 min., 58 degrees C for 1 min., and 72 degrees C for 1 min., and a final elongation step at 72 degrees C for 10 min. Amplified products were analysed by electrophoresis on 2% agarose gels. Results and Discussion The histology of the retinas of C3H/HeJ and C3H/RV mice is shown in Figure 1. In the C3H/RV strain three well developed cellular layers including the intact photoreceptor rod cell layer were seen in all individuals (Fig. lA), whereas the layer of photoreceptor rod cells was absent in all C3H/HeJ mice (Fig. lB), confirming other reports of the presence of the mutated rd allele in this strain. In further studies the retinas of C57BL/6J and BALB/c mice were found to be histologically normal while in SJL the photoreceptor rod cells were absent (data not shown), in agreement with other observations. The Southern blot analysis using a cDNA probe for the rd locus (10) suggested that the histological difference in the retinal tissue of the congenic C3H strains was due to structural variants at this genomic locus (Fig. 2). The presence of normal retinas in C3H/RV mice coincided with the existence of 4.0, 1.9 and 0.7 kb Msp I polymorphic DNA fragments which were absent in the digested DNA from mice of the C3H/HeJ strain (Fig. 2). Although the allelic differences at the Ric and Flv loci in C3H/HeJ and C3H/RV mice have already been recognised, this is the first report of an allelic difference at the rd locus in these congenic strains. These data strongly support the coinheritance of the Ricr and rd wild type alleles with the selected Flvr allele from the PRI donor mouse strain into the genetic background of C3H/He mice. Although the PRI strain is no longer available for confirmatory studies, the evidence indicates that chromosome 5 is the likely location of the Flv locus. To assist with the mapping of Flv, the Beta-Glucuronidase (Gus) locus has been chosen as a potential candidate for a distal anchor locus on chromosome 5. Although this locus has been characterised in many strains of laboratory mice, the allele carried by C3H/RV mice has not been described. In order to analyse this locus in C3H/HeJ, C3H/RV and other strains we have developed a rapid and accurate method based on PCR amplification of the polymorphic region in intron 4 of the Beta-glucuronidase structural gene (11). The method exploits the insertion of the 206-bp B2c repetitive element into the Gus-sa allele of BALB/c mice which is absent from the Gus-sb allele of C57BL/6J mice (12). PCR amplification produced a 283 bp fragment from BALB/c DNA, but only a 77 bp fragment from C3H/HeJ, C3H/RV and C57BL/6J DNA (Fig. 3). Although this approach did not distinguish between the Gus-sh and Gus-sb alleles (Fig. 3), it is likely that C3H/RV mice carry the Gus-sh allele of their C3H/He progenitors. In conclusion, this study has identified an allelic difference between the congenic strains C3H/HeJ and C3H.PRI-Flvr (C3H/RV) at the rd locus, indicating that the Flv locus is linked to Ric and rd on chromosome 5. We have also shown that a PCR-based method could be used for detecting allelic forms of the anchor locus Gus-s in future studies to map the position of the Flv locus. References 1. Sabin, A.B. Ann. N.Y. Acad Sci 54: 936-944 (1952) 2. Sangster, M.Y., Heliams, D.B., Mackenzie, J.S. and Shellam, G.R. J Virol 67: 340-347 (1993) 3. Green, M.C. In Genetic variants and strains of the laboratory mouse (M.F. Lyon and A.G. Searle, ed.), pp. 124-125, Gustav Fischer Verlag, Stuttgart (1989). 4. Groschel, D. and Koprowski, H. Arch Gesamte Virusforsch 17: 379-391 (1965) 5. Groves, M.G., Rosenstreich, D.L., Taylor, B.A. and Osterman, J.V. J Immunol 125: 1395-1399 (1980). 6. Jerrells, T.R. and Osterman, J.V. Infect Immun 31: 1014-1022 (1981). 7. Lyon, M.F. and Kirby, M.C. Mouse Genome 91: 40-80. (1993). 8. La Vail, M.M. and Sidman, R.L. Arch Ophthalmol 91: 394-400 (1974). 9. Patarca, R., Freeman, G.J., Singh, R.P., Wei, F-Y, Durfee, T., Blattner, F., Regnier, D.C., Kozak, C.A., Mock, B.A,, Morse, H.C., Jerrells, T.R. and Cantor, H.J. Exp Med 170: 145-161 (1989). 11. D'Amore, M.A., Gallagher, P.M., Korfhagen, T.R. and Ganschow, R.E. Biochem 27: 7131-7140 (1988). 12. Gallagher, P.M., D'Amore, M.A., Lund, S.D., Elliot, R.W, Pazik, J., Hohman, C., Korfhagen, T.R. and Ganschow, R.E. Genomics 1: 145-152 (1987). FIGURE 1. (Legend). Retinal histology of congenic C3H/RV and C3H/HeJ mice (x25). (A) The normal retina of C3H/RV mice shows the presence of all three cellular layers: rod photoreceptor cell (RPC), bipolar neuron (BN) and ganglion cell (GC) layers. (B) The degenerated retina of C3H/HeJ mice lacks the RPC layer. FIGURE 2. (Legend). Southern blot analysis of the rd locus. Genomic DNA from individual C3H/RV (1-4) and C3H/HeJ mice (5-8), digested with Msp I, was hybridised to the rd cDNA probe. Molecular weight standards are shown on the right. Arrows at left indicate the polymorphic DNA fragments present only in the C3H/RV DNA. FIGURE 3. (Legend). PCR analysis of the Gus locus in different mouse strains. The polymorphic region of the Gus-s gene in C3H/HeJ [HeJ]; (C3H/HeJxC3H/RV)Fl [HeJxRV]; C3H/RV [RV]; (BALB/cxC3H/RV)F1 [Balb/cxRV]; BALB/c; (C57BL/6JxC3H/RV)F1 [B6xRV] and C57BL/6J [B6] was amplified by PCR and analysed on 2% agarose gels. Molecular weights of DNA standards are shown at left and of the two polymorphic fragments produced at right. |
