| First Author | Wallace ME | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 3 | Pages | 416-20 |
| Mgi Jnum | J:2536 | Mgi Id | MGI:51058 |
| Citation | Wallace ME (1992) Embryonal cells of Peruvian mice have no evident defect in DNA repair. Mouse Genome 90(3):416-20 |
| abstractText | Full text of Mouse Genome contribution: EMBRYONAL CELLS OF PERUVIAN MICE HAVE NO EVIDENT DEFECT IN DNA REPAIR. Two suggestions have been made as to the cause of the high spontaneous incidence of mutants and of chromosomal abnormality shown by the wild population of Peruvian mice; these are that it carries a genetic agent, which destabilises transposable elements, and that it is defective in DNA repair mechanisms (Wallace and Nash,7 Wallace5). Possibly the latter question may simply be answered by examining the uv survival curves of an easily repairable DNA virus like herpes simplex virus in cells derived from this line of mice. We irradiated virus under standard conditions with various doses of uv light and then titrated the surviving infectivity in several cell lines in parallel. Other work (Lytle,3 Cameron et al.l) has shown that cell lines vary greatly in their apparent ability to repair lesions in herpes virus DNA (Fig. 1). Mouse embryo cells from several sources were therefore activated and tested for their ability to support the replication of unirradiated and irradiated virus. Two experiments were done. In the first, 10 embryos derived from two Peruvian mothers were individually crudely screened. In the second, four embryos derived from one Peruvian mother were used for finer quantitative studies. In the first, the Peruvian embryos came from two daughters mated to their father; in the second from one daughter mated to her father. The two fathers were members of a selection line set up from descendants of mice trapped in site 1 of the Peruvian population (Wallace and Lowe,6 and Wallace5). In this line, selection had been exercised in favour of animals with spontaneous chromosome abnormalities. The first father had one cell, out of 100 scored, with a translocation between the X chromosome and an autosome, and the second had, out of 100 cells scored, one cell with an autosomal translocation, and four cells each showing a chromosome fragment separated from the normal complement of 20 chromosomes. The embryos were thus highly likely to be mosaic for chromosome abnormalities, like their fathers. In both experiments each embryo was handled separately and taken 16 days after conception. The trypsinised cells were cultured and passaged until sufficient were available (3 to 7 passages). The cells varied greatly in their cultural characteristics for reasons that have not been determined. Similar methods were used to derive cells from other lines of mice. The screening experiments involved the irradiation of a single suspension of herpes simplex (type 1) virus (strain SC 16) under standard condition (Ross et al.4). Aliquots of each suspension were stored at Ð70 degrees C until required. The source of uv light was a Hanovia 30 watt low-pressure mercury discharge lamp in quartz which emits energy principally at 254 nm. The incident dose rate was determined using potassium ferrioxalate actinometry (Hatchard and Parker2). Cell lines from each embryo were plated out in 5 cm Petri dishes using Eagle's (Glasgow) medium containing 10% tryptose phosphate broth (Difco) and 10% fetal calf serum. When confluent the monolayers were used to assay the sorted virus by a standard plaquing method. For comparison cell lines derived from other strains of mice were tested as well as BHK 21 cells. The survival curves of infectivity of virus titrated in these independently derived cell cultures from Peruvian mice are shown in Figure 1 along with those of other cell lines. As expected all mouse lines showed reduced survival of irradiated virus at the doses tested when compared with BHK cells. In these experiments cells derived from Balb/c mice seemed more able to repair irradiated virus than other mouse strains, but this impression may be misleading. It was clear (a) that the mouse strains tested here did not behave much differently from the cells of the 'Lister Institute' mice previously tested (Cameron et al.1), and (b) that Peruvian mouse embryo cells behaved homogeneously. One of the benefits of this approach is that not only can the survival of irradiated virus be estimated but the size of the resulting plaques may also be measured. The small plaques previously noted with irradiated herpes virus arise because there is a 'repair delay' in the cell initiating each plaque. This delay which occupies several hours relates to the capability of the cell to repair DNA (Ross et al.4). The sizes of plaques obtained in the above experiments were therefore measured. The image of the plaques were projected using a magic lantern at standard magnification and the sizes of 25 plaques were estimated. From these measurements the areas of plaques were determined, as also the median area. The result (Fig. 2) is clear. Plaques certainly did become diminished in size in monolayers of Peruvian mouse cells after irradiation of virus, strongly supporting the view that repair processes were active. In the second experiment the D37 dose was estimated in the following way. Virus (SC16) was irradiated at a dose-rate of 3.6 ergs/mm2/sec. The time intervals chosen were 0, 10, 20, 30, 40, 60, 120 seconds (=x). The surviving virus was assayed at each dose and expressed as log10 pfu.ml (=y). This was done using several cell lines including four independently derived lines from four Peruvian mouse embryos and from embryos of Balb/c, B10a and CBA mice; BHK cells were included for comparison. Because survival curves tend to curve upwards at higher uv doses, those points where there was less than 1/30 survival have been disregarded. The remaining data have been used to derive a regression of the first kind where dose (x) is regarded as invariate. Thus for each cell-line equation y = mx + c was obtained using the least squares method. From the value m (i.e. the slope of the regression) the D37 dose was derived (see Table 1). The correlation coefficients obtained for the various cell lines show that the regressions derived are reasonable, and it is clear from Table 1 that the four independent cell isolates from Peruvian mice repaired uv-damaged herpes virus genomes quite as efficiently as did other mouse cells. These mice must therefore be regarded as normal in this respect. Interestingly the D37 values obtained here for BHK cells are about one third of what others (including ourselves) have published while those for mouse cells are in closer agreement. It is concluded that Peruvian mouse cells do not differ essentially from other mouse cells in their ability to repair irradiated virus. In consequence, the frequency of chromosomal abnormality observed in these mice cannot be accounted for on the grounds of a deficient DNA repair system. References 1. Cameron, K.R., L.M. Tomkins, R.P. Eglin, L.J.N. Ross, P. Wildy and W.C. Russell. The effects of ultraviolet and ionizing radiations on herpes viruses, SV40 and adenoviruses in relation to the small-plaque effect. Arch. Virol., 62:31-40 (1979). 2. Hatchard, C.G. and C.A. Parker. A new sensitive actinometer. II. Potassium ferrioxalate as a standard chemical actinometer. Proc. Roy. Soc., London., A235, 518-536 (1956). 3. Lytle, C.D., host cell reactivation in mammalian cells. I. Survival of ultra-violet irradiated herpes virus in different cell lines. Int. J. Rad. Biol., 19, 329-337 (1971). 4. Ross, L.J.N., K.R. Cameron and P. Wildy. Ultra-violet irradiation of herpes simplex virus: reactivation processes and delay in virus multiplication. J. Gen. Virol., 16, 229-311 (1972). 5. Wallace, M.E., An inherited agent of mutation and chromosome damage in wild mice. J. Hered. 76, 271-278 (1985). 6. Wallace, M.E. and C.A. Lowe, Selection in Peru Mice. Mouse Newsletter, 71, 18 (1984). 7. Wallace, M.E. and H.R. Nash A tiger mouse and relatives - variants caused by an activated transposable element? J. Hered., 75, 2-7 (1984). TABLE 1 Cell line: Peruvian mice: C1; ÔmÕ (slope:log10y/x sec): -0.034; ÔrÕ: -0.78; D37: seconds: 12.70; ergs mm2: 45.20. Cell line: Peruvian mice: C2; ÔmÕ (slope:log10y/x sec): -0.036; ÔrÕ: -0.92; D37: seconds: 13.38; ergs mm2: 48.18. Cell line: Peruvian mice: C3; ÔmÕ (slope:log10y/x sec): -0.040; ÔrÕ: -0.87; D37: seconds: 12.05; ergs mm2: 43.38. Cell line: Peruvian mice: C4; ÔmÕ (slope:log10y/x sec): -0.043; ÔrÕ: -0.95; D37: seconds: 10.04; ergs mm2: 36.15. Cell line: Standard inbred lines: B10; ÔmÕ (slope:log10y/x sec): -0.046; ÔrÕ: -0.93; D37: seconds: 9.39; ergs mm2: 33.73. Cell line: Standard inbred lines: Balb/c; ÔmÕ (slope:log10y/x sec): -0.040; ÔrÕ: -0.92; D37: seconds: 10.80; ergs mm2: 38.86. Cell line: Standard inbred lines: CBA; ÔmÕ (slope:log10y/x sec): -0.037; ÔrÕ: -0.89; D37: seconds: 13.02; ergs mm2: 46.88. Cell line: Standard laboratory cell line (BHK); ÔmÕ (slope:log10y/x sec): -0.024; ÔrÕ: -0.97; D37: seconds: 18.00; ergs mm2: 64.77. Cell line: Standard laboratory cell line (BHK); ÔmÕ (slope:log10y/x sec): -0.020; ÔrÕ: -0.85; D37: seconds: 21.50; ergs mm2: 81.70. D37 doses calculated from uv-survival data for herpes virus assayed on various cell lines. Four independently derived Peruvian mouse-embryo cell lines (C1-C4) behaved homogeneously showing similar rates of inactivation. These rates were also similar to those of other mouse cell lines (B10, Balb/c and CBA). All mouse-cell lines were less able to repair virus infectivity than BHK cells (p<0.001). LEGENDS Figure 1. UV-survival curves in various cell lines. The dashed curves are derived from previously reported data for BS-C1, BHK-21 and ME cells (Cameron et al., 1979). These clearly indicate that survival of infectivity varies very considerably according to the cells in which titrations are carried out. They reflect the ability of the lines to repair DNA (Host-cell repair: dark repair). The points are derived from a single experiment in which four samples of herpes simplex virus (SC16) were titrated after uv irradiation under standard conditions with 0,433,866 and 1732 ergs/mm2. The titrations were made in BHK-21 cells (3 independent experiments); Peruvian mouse cells (10 experiments with 10 independently derived lines); a B10 cell line, a CBA cell line, and a Balb/c cell line. The bars indicate one standard deviation calculated on the geometric mean. Figure 2. The plaque sizes of unirradiated and irradiated herpes virus (SC16). The plaques arising from the experiments shown in Fig. 1 were measured at standard magnification when projected in a magic lantern. Twenty-five plaques were recorded for each determination. The standard magnification used was 10 x. Plaques were outlined on metric graph paper and the mm squares inside each outline were counted. Frequency distribution curves were examined by probit analysis and the points represent median plaque sizes. Clearly, the five Peruvian mouse cell lines showed similar reductions in the size of plaques as did Balb/c line and BHK-21. Margaret E. Wallace, the late P. Wildy and P. Rebello; Department of Pathology, University of Cambridge, Cambridge, England. |
