| First Author | Moore TF | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 4 | Pages | 687-89 |
| Mgi Jnum | J:3478 | Mgi Id | MGI:51991 |
| Citation | Moore TF, et al. (1992) Failure to detect age-related reactivation at the Hprt locus. Mouse Genome 90(4):687-89 |
| abstractText | Full text of Mouse Genome contribution: FAILURE TO DETECT AGE-RELATED REACTIVATION AT THE Hprt LOCUS. T. F. Moore1, SA. Sheardown2 and D.G. Whittingham. MRC Experimental Embryology and Teratology Unit, St. George's Hospital Medical School, Cranmer Tce., London SW17 ORE, UK. 1Author for correspondence. 2Current address: Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, UK. Introduction Age-related instability of the maintenance of X-chromosome inactivation has been demonstrated at the widely separated sparse fur (spf) and mottled (Moblo) loci(1, 2), suggesting that there is a general destabilization of X-inactivation with increasing age. However, a subsequent study failed to detect reactivation at the tabby (Ta) locus(3), arguing that certain loci which map close to Xce may be more resistant to reactivation. At present it is unclear whether age-related reactivation of X-linked genes is limited to certain loci or whether it occurs on a chromosome-wide basis due to sequential reversal of the maintenance of X-inactivation along the X-chromosome. It is also unknown whether there are tissue or species differences in reactivation at a particular locus. It has been argued that species differences in reactivation may occur due to the possibility of differences in the rigour of maintenance of CpG methylation4. For a number of reasons the mouse Hprt locus is particularly suited to addressing these questions. First, its location is roughly equidistant between the spf and Ta loci on the X-chromosome. Second, it encodes a constitutively expressed enzyme, HPRT, allowing a wide range of tissues to be examined for reactivation. Third, since in a previous study reactivation of the human HPRT gene was not detected5, it provides an opportunity to compare the behaviour of this locus in a long-lived and in a short-lived species and thereby a means of testing Holliday's proposal(4) concerning the possibility of species differences in age- related gene reactivation. The use of the T16H mutant to study age-related reactivation at X linked loci has been described previously(1, 2). Results Production of T16H Hprt(b-m3) +/+ + Ta females. This study was facilitated by the availability of the HPRT-deficient mouse mutant, Hprt(b-m3) produced by the ES cell method(6). The Hprt(b-m3) null allele contains a large deletion which abolishes transcription at the Hprt locus(7, 8) and produces a characteristic RFLP when DNA is digested with BamH1(7). Female mice of genotype T16H +/+ Ta, and Mo(bio)/Y males were obtained from the MRC Radiobiology Unit (Division of Genetics), Harwell. The T16H genotype was maintained as described(9). Females of genotype T16H Hprt(b-m3) +/+ + Ta were produced as follows: T16H +/+ Ta females were mated to Hprt(b-m3)/Y males, and HPRT-positive agouti female offspring (putative T16H +/+ Hprt(b-m3) were mated to Ta/Y males. Two HPRT-deficient agouti females from this cross (putative T16H Hprt(b-m3) +/+ + Ta) were test-mated to Mo(bio)/Y males to confirm the presence of the desired genotype. Both females produced offspring consistent with the presence of the T16H Hprt(b-m3)3 + /+ + Ta genotype. RFLP analysis of putative T16H Hprt(b-m3) +/+ + Ta females. To confirm the T16H Hprt(b-m3) +/+ + Ta genotype in stock females used in this experiment, DNA was extracted from tail biopsies, digested with BamHl, fractionated on 1% agarose gels, blotted on to Hybond N + membrane (Amersham, UK), and hybridized to the Pst1 insert of the plasmid pHPT5 (ref.10) as described(11). All of the tested females displayed the heterozygous RFLP pattern. This, combined with the finding of a HPRT-deficient phenotype following tail-bleeding and HPRT assay, confirmed the presence of the T16H Hprt(b-m3) +/+ + Ta genotype in these females. HPRT activity in tissues from T16H Hprt(b-m3) +/+ + Ta females. Mice were killed by CO2 inhalation and tissue samples were collected and stored at Ð70 degrees C until used. For assay, samples were thawed, disrupted in a 1 ml Wheaton homogenizer, and assayed immediately as described(12). HPRT activity was measured in triplicated samples of blood lysates, and 10,000 rcf supernatants of cerebellum, liver, spleen, tongue, ovary, and kidney homogenates. Samples were incubated at 37 degrees C for 4-5 h. Protein concentration of supernatants was measured in duplicate by the method of Lowry(13). The sensitivity of the assay varied according to the specific activity of the tissues, but was estimated to be capable of detecting 0.05-0.1% of normal activity in supernatants of all tissues, except blood lysates, in which the limit of detection was 0.5% of normal activity. The results are summarized in the Table. The data were combined according to age of mice as follows: group 1) 3-4 months (n=5); 2) 12-14 months (n=4); and 3) 18-20 months (n=7). Positive and negative controls consisted of tissues from Hprt(b)/Hprt(b) (n=3) and Hprt(b-m3)/Hprt(b-m3) (n = 4) adult females, respectively. Square root transformation of the data was carried out prior to analysis by oneway analysis of variance. Significant differences in HPRT activity were not detected between the three age groups in any of the tissues examined (p>0.05 in all cases). The data in the three groups were also compared using Anova, on the combined HPRT activity in all tissues. Again, no correlation between age and HPRT activity was detected (F(2) <3.134, in all cases). Discussion Contrary to a previous prediction(4), significant age-related reactivation at the mouse Hprt locus was not detected in this study. A suggestion that the reactivation of previously silenced genes may be tissue-specific, or display inter-species differences(5) is not supported by this study, but does not preclude such effects at other loci. The failure to detect age-related reactivation at the Hprt locus means that 50% of loci examined in the mouse do not reactivate (Ta and Hprt), and 50% do (spf and Mo(bio)). The relative positions of these loci in relation to Xce suggest that the behaviour of a particular locus may be intrinsic to that locus and not due to its chromosomal position per se. It is difficult to compare the sensitivity of the method used in this study to other studies of age-related reactivation because of the wide variety of techniques used. A highly significant (fifty-fold) increase in the reactivation rate of the spf gene in old mice, producing a positively stained area of 2.8% on histological sections, was detected in a previous study(1). Such a degree of reactivation at the Hprt locus would have been well within the sensitivity of the HPRT assay used here. Lack of detectable HPRT activity in the ovaries of young (3-4 months) T16H Hprt(b-m3) +/+ + Ta females was an unexpected finding because reactivation of the Hprt(b) allele on the normal X-chromosome is expected to occur in meiotic oocytcs of this genotype. This observation is similar to that of a previous study(14) in which apparently incomplete reactivation of the Pgk-1(a) allele may have been observed in meiotic oocytes from TI6 Pgk-1(b)/+ Pgk-1(a) females, and supports a postulated mechanism of Xce-linked meiotic drive in which the inactive X may not undergo full functional reactivation on entry of oocytes into meiosis(8). However, it is possible that the HPRT assay used in this study was not of sufficient sensitivity to detect the relatively small proportion of oocyte HPRT which may be present in whole ovary supernatants. Comparison of HPRT activity in purified oocytcs from T16H Hprt(b-m3)/+ + and T16H +/+ Hprt(b-m3) females will be used to resolve this question in future experiments. HPRT activity in tissues from T16H Hprt(b-m3) +/+ + Ta females. Tissue: Cerebellum: Age: 3-4 months (n = 5): 0.011 +/- 0.010 (0.001-0.026); Age: 12-14 months (n = 4): 0.008 +/- 0.009 (0.000-0.018); Age: 18-20 months (n = 7): 0.043 +/- 0.060 (0.000-0.163); Positive Control (n = 3): 6.369 +/- 4.764 (3.320-11.860). Tissue: Tongue: Age: 3-4 months (n = 5): 0.005 +/- 0.008 (0.000-0.017); Age: 12-14 months (n = 4): 0.023 +/- 0.045 (0.000-0.090); Age: 18-20 months (n = 7): 0.017 +/- 0.021 (0.000-0.053); Positive Control (n = 3): 2.876 +/- 1.952 (0.630-4.171). Tissue: Liver: Age: 3-4 months (n = 5): 0.016 +/- 0.019 (0.000-0.046); Age: 12-14 months (n = 4): 0.043 +/- 0.054 (0.000-0.111); Age: 18-20 months (n = 7): 0.088 +/- 0.218 (0.000-0.580); Positive Control (n = 3): 2.897 +/- 0.904 (1.896-3.655). Tissue: Spleen: Age: 3-4 months (n = 5): 0.008 +/- 0.018 (0.000-0.040); Age: 12-14 months (n = 4): 0.069 +/- 0.062 (0.023-0.159); Age: 18-20 months (n = 7): 0.003 +/- 0.008 (0.000-0.022); Positive Control (n = 3): 5.399 +/- 1.044 (4.200-6.107). Tissue: Blood: Age: 3-4 months (n = 5): ND; Age: 12-14 months (n = 4): ND; Age: 18-20 months (n = 7): 0.000 +/- 0.001 (0.000-0.002); Positive Control (n = 3): 0.536 +/- 0.268 (0.240-0.763). Tissue: Ovary: Age: 3-4 months (n = 5): ND; Age: 12-14 months (n = 4): ND; Age: 18-20 months (n = 7): 0.001 +/- 0.002 (0.000-0.005); Positive Control (n = 3): 15.290 +/- 3.748 (11.219-18.600). Tissue: Kidney: Age: 3-4 months (n = 5): ND; Age: 12-14 months (n = 4): ND; Age: 18-20 months (n = 7): ND; Positive Control (n = 3): 3.155 +/- 1.957 (1.811-5.400). HPRT activity is expressed in nmoles h(-1) mg protein(-1) (mean +/- SD). Figures in parenthesis are range of sample values per age group. ND, activity not detected. References 1. Wareham, K.A., Lyon, M.F., Glenister, P.H. and Williams, E.D. (1987) Nat. 327:725-727. 2. Brown, S. and Rastan, S. (1988) Genet. Res., Camb. 52:151-154. 3. Cattanach, B.M. (1991) Mouse Genome 89(1):272. 4. Holliday, R. (1989) Nat. 337:331. 5. Migeon, B.R., Axelman, J. and Beggs, A.H. (1988) Nat. 335:93-96. 6. Hooper, M.L., Hardy, K., Handyside, A., Hunter, S. and Monk, M. (1987) Nat. 326:292-295. 7. Thompson, S., Clarke, A.R., POW, A.M., Hooper, M.L. and Melton, D.W. (1989) Cell 56:313-321. 8. Moore, T.F. (1992) PhD Thesis, Univ. of London. 9. McMahon, A. and Monk, M. (1983) Genet. Res., Camb. 41:69-83. 10. Konecki, D.S., Brennand, J., Fuscoe, J.C, Caskey, C.T. and Chinault, A.C. (1982) Nucl. Acids Res. 10: 6763-6775. 11. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Cold Spring Harbour Press, USA. 12. Monk, M. (1987) Mammalian Development - A practical approach, IRL Press, UK. 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. (1951) J. Biol. Chem. 193:265-275. 14. Johnston, P.G. (1981) Genet. Res., Camb. 37:317-322. This work was supported by an Action Research Training Fellowship to T.F. Moore. The invaluable assistance of A. Millhouse and P. Glenister is acknowledged. |
