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Tests for recombinagens in mammals in vivo
Mutation Research, 284 (1992) 177-183
177
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 00383
Tests for recombinagens in mammals in vivo Rudolf Fahrig Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Abteilung Genetik, Hannover, Germany (Accepted 30 March 1992)
Keywords: Reciprocal recombination; Heterozygous recessive mutations; Spot test
History In rare instances heterozygous animals show a patch of fur for the recessive phenotype, and then the gene has definitely affected the coat. Recombinational processes rather than gene mutations belong to the most potent mechanisms for recessive genes to come to expression. Justification for this statement comes from experiments using yeast, in which the probability that a heterozygous recessive gene becomes homozygous is two orders of magnitude higher for nonreciprocal recombination than for gene mutation (Fahrig, 1976). Also, observations in cultured mouse cells showed that the frequency of nonreciprocal recombination (gene conversion) between repetitive genes is several orders of magnitude higher than the frequency of gene mutation (Liskay and Stachelek, 1983). With reciprocal recombination a single event is sufficient to result in the expression of all recessive mutations of a chromosomal segment, whereas with gene mutations several single events would be needed in order to achieve a similar effect. In spite of that, as possible mechanisms in mammals they have not often been considered. The reason for this is that mitotic chromosome pairing has not been observed in mammals.
Correspondence: Dr. R. Fahrig, Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Abteilung Genetik, Nikolai-Fuchs-Str. 1, D-3000 Hannover 61, Germany.
Nevertheless, 5 years before Stern's (1936) classical work with Drosophila, Keeler (1931) considered the possibility of mitotic crossing-over, i.e., reciprocal recombination in mice. But shying away from this idea, he proceeded to give a different interpretation of his data. Therefore, the first case with convincing evidence for mitotic reciprocal recombination in mammals as mechanism excluding somatic mutation as a possible cause was (Carter, 1952) a mosaic mouse born in 1948 in the first of her parents' three litters. Her father was of the wild-type CBA inbred strain; her mother was a cross-bred heterozygote for the semidominant color and macrocytic anemia mutant W v. The mosaic showed, over the greater part of her body, the characteristic phenotype of a W V / + heterozygote; there was a white brown spot, a large white belly patch, the agouti hairs of the dorsum were bleached and the ventral surface was silvered. Two regions, however, showed full, wild-type coloration. The greater part of the wild-type coloration was on the right side of the mouse, and this position suggested that the right gonad, or possibly both, might have been involved; the mosaic was therefore mated to a wild-type male to test the segregation of W v. Of the 41classified young, 10 were wild-type and 31 were typical W V / + heterozygotes; the deviation from the expected 1 : 1 ratio was significant statistically (P = 0.00145). None showed any sign of mosaicism. The data established that the mosaic female developed from a W V / + zygote, that parts of her
178
soma were deficient of W v, and that she bred as though her germinal tissues were partly deficient of +w. Somatic mutation could hardly have been the mechanism of formation, since two mutational steps would have to be postulated, W v to + in the soma and + to W v in the gonad. Deletion likewise fails to provide an explanation, since two deletions, in homologous chromosomes, would have to be postulated. The only possible explanation is given by mitotic recombination. The W" mosaic mouse would be supposed to have arisen by crossing-over proximal to the locus of W v, at an early cleavage division. The daughter cells would have the constitutions W v / w v and + / + and would have given rise to one ovary and to the fully colored patches respectively. The affected ovary would produce only WV-bearing ova; the unaffected ovary would produce W v- and +-bearing ova in equal numbers; and the expected segregation ratio would again be 3 : 1, agreeing well with observation (Fig. 1). Grfineberg (1966) extracted material from the literature to show that mosaicism in mammals is best explicable by mitotic crossing-over and not by mutation. He discussed nearly all published cases of mosaicism in mice (Pincus, 1929; Fisher, 1931; Feldman, 1935; Morgan and Holman, 1955; Russell and Major, 1957), rats (Castle, 1922; Curtis and Dunning, 1940), rabbits (Castle, 1922; Pickard, 1936) and guinea pigs (Wright and Eaton, 1926). But in conclusion he had to admit that also other mechanisms than recombination due to crossing-over have to be considered for expres-
Gonads
Soma
Fig. 1. Interpretation of Carter's case in terms of mitotic crossing-over.
b !Oiiii~i~iiiiii!ilililililiiii!
deletion or • reciprocal b
chromosome loss
I
b
b
+
b
recombination
I
nonreciprocal ~r
b
gene mutation
recombination b
b Fig. 2. Different ways for a heterozygous recessive mutation to come to expression.
sion of the recessive genes. Fig. 2 shows that other possible mechanisms are gene mutation, chromosome loss, non-disjunction, chromosome deletion, or nonreciprocal recombination due to gene conversion * As an addendum to Grfineberg's extensive survey of evidence for mitotic crossing-over in mammals (1966), Bateman (1967) published a short note. In contrast to the case of Carter (1952), the case which was the subject of this note was un-
There are two distinct possible genetic consequences of a recombinational interaction. They are known as reciprocal recombination due to crossing-over and non reciprocal recombination due to gene conversion. Crossing-over is seen as a reciprocal breakage and rejoining of homologous molecules such that all information on one side of the crossing-over is recombined reciprocally with all information on the other side. Mitotic crossing-over is detected as homozygosis for all markers distal to the crossing-over. Mitotic crossing-over can lead to homozygosis only when it occurs in G2 (the four strand stage). Mitotic gene conversion can occur in GI and is defined as a nonreciprocal transfer of information. This type of mitotic recombination is a repair process which is known to repair double strand breaks and to fill double strand gaps by copying a homologous sequence. The words crossing-over and conversion are used to denote not only the genetic outcome of these recombinational processes but also the processes themselves. (Hastings, 1988)
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doubtedly somatic in origin. Mitotic crossing-over differs from all other phenomena which may have an outward resemblance to it, by producing two complementary genotypes, though one of these is usually indistinguishable phenotypically from the heterozygous state from which it arose. In the case described by Bateman (1967) both homozygotes were distinguishable from the heterozygote. The mouse was non-agouti and heterozygous for c ch (chinchilla) and c (albino). The heterozygote is fawn-colored (light brown) and the homozygotes are dark gray, also called sepia (cch/cch), and white (c/c). Somatic crossing-over between the c-locus and the centromere had produced just such a twin-spotting of dark gray and white as could be expected theoretically. Present
Fisher et al. (1986) have made an attempt to distinguish between reciprocal recombination and somatic mutation or non-disjunction in mice. But they found little evidence for mitotic recombination following X-irradiation of 10.25-day-old embryos heterozygous in repulsion for pink-eyed dilution (p) and albino (c), two linked loci assigned to chromosome 7. The reason for not having succeeded was that for lack of microscopical hair analysis, the authors could not address the question as to whether the presence of white spots was due to genetic alterations expressing the albino genotype or to pigment cell death. As nearly all spots observed by them were white, an interpretation of results was not possible at all. A more convincing report about spontaneous and induced mitotic recombination comes from Panthier et al. (1990). In this report, phenotypic reversions of the W ei allele at the W locus were studied. Mice heterozygous in repulsion for both W ei and buff (bf) (i.e., W ei + / + bf) were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khakicolored coat of nonagouti buff homozygotes ( a / a : bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a / a : W ei + / + b f mice: either solid black or khaki-colored.
Furthermore phenotypic reversions o f w e i / q were enhanced significantly following X-irradiation of 9.25-day-old W e i / + embryos ( P < 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. Twin spots with w e i + / w e i + and + b f / + bf genotypes respectively would have made a much stronger case for mitotic recombination. No such twin spots were observed by Panthier et al. (1990). However, they were probably not observable because all available evidence indicates that death of melanoblasts is responsible for the white spotting of the coats of W locus mutants (Silvers, 1979). Accordingly; a W ei q- / W ei q- homozygous clone would be expected to die, leaving an area which might be colonized or not by W e~ + / + bf or + b f / + bf surrounding melanoblasts. Similarly a single mitotic crossing-over between the W locus and the bf locus would be expected to produce a clone of w e i b f / + bf melanocytes. Again no such clones were observed. This presumably results from the inability to detect w e i b f / + bf patches, composed of white hairs interspersed among khaki rather than black hairs, on the coat of W ei + / + bf mice. It has recently become possible to detect mitotic reciprocal recombination at the dilute locus of mice in vivo by using methods of molecular biology. The dilute locus has served as a useful visual marker in a number of studies designed to measure mutation rates in the mouse following X-ray or chemical treatment. For example, in a study of Russell (1971), 235 radiation-induced mutations involving the dilute locus and a closely linked marker, short ears (within 0.1 cM), were identified. Complementation tests revealed 16 different complementation groups. Four classes of forward mutations to dilute were identified including dilute, viable ('d'); dark dilute, viable (d*); dilute, opisthotonic (d°P); and prenatally lethal (dPJ). As these mutations are X-ray-induced, many of them may represent deletions or DNA rearrangements. In some cases, dilute mutations were identified that included both dilute and short ears and thus probably represent large deletions. At present, the dilute (d v) coat color mutation of DBA mice provides the only simple means for
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measuring the relative somatic and germ-line reverse mutation rates of retrovirus-induced mutations in mammals. The d v mutation was generated by the spontaneous integration of an ecotropic murine leukemia virus into noncoding sequences of the dilute locus. Reversion of the d v mutation occurs by provirus excision and is mediated by homologous recombination events involving the viral long terminal repeat sequences. Although numerous independent germ-line d + revertants have been identified, only one case of somatic d + rcvertants has been reported. Over 5 years, Seperack et al. (1988) have screened more than one million mice homozygous for the d v mutation to determine whether they could identify somatic d v revertants. This survey has resulted in the identification of one somatic d + revertant. By Southern blot analysis of EcoRI-digested DNA from the somatic revertant, the authors were able to show that the revertant examined appeared to be the result of reciprocal recombination between homologous chromosomes. Test system Instead of checking one million mice for spontaneously occurring color spots, it is more sensible to induce such spots by treatment with chemical mutagens or even X-rays, which were used by Russell and Major (1957) in their pioneer experiments. The only suitable test system to detect reciprocal recombinations is the spot test with mice (Fahrig, 1975). According to this method mouse embryos, which are heterozygous for different recessive coat color genes, are treated in utero
between the 9th and 1 lth day post conception by injection of a mutagen into the peritoneal cavity of the mother animal, or by other appropriate routes of administration. If this treatment leads to an alteration or loss of a specific wild-type allele in a pigment precursor cell, a color spot in the coat of the adult animal may appear. With regard to the mechanism (see Fig. 2) by which the heterozygous recessive coat color alleles can be expressed, this is either a gene mutation, theoretically also loss of the wild-type allele through deletion or monosomy, or a recombinational process such as mitotic crossing-over (reciprocal recombination), or mitotic gene conversion (nonreciprocal recombination). Of the numerical and structural chromosome aberrations that can lead to loss of the wild-type allele, only those that survive several mitoses would cause a spot with expression of the recessive allele. In the course of the routinely performed spot test, three types of spots are distinguished: (1) white midventral spots (which have no pigment at all), regarded as resulting from pigment cell killing; (2) spots with hairs similar to the yellow hairs which normally surround ears, genital papillae, and mammae, classified as misdifferentiation spots and appearing as yellow fluorescent hairs with agouti genotype; (3) spots of genetic relevance (SGR) resulting from genetic alterations at the different gene loci (Table 1) and expressing the recessive mutant or their wild-type alleles. It is apparent that without routinely performed microscopical analysis it is not possible to distinguish between the different mechanisms leading to expression of a recessive mutation. Therefore, nearly all published work about the spot test cannot be used for the specific subject of this
TABLE 1 THEORETICAL EXPECTIONS Original state
b/B
Theoretically color spots can be induced by Mutation (a) b,/b ~ Deletion ( J ) b/zl Monosomy (0) b/0 Reciprocal recombination b/b Nonreciprocal recombination (*) b/b* Color of spot Brown
p/P
d/D
cch/c
p/p~ p/3 p/p p/p* Light gray
d/da d/A d/0 d/d d/d* Gray
cCh/c a cCh/A Light brown
p cCh/p C
(p cCh/A) (p cch/0) -p c c h / p c ch twin spot
(P C / A ) (P C / 0 ) PC/PC
Near-white
Maternal black
181
review. The only possibility to distinguish between induced mutations and induced reciprocal recombinations is to use specific mouse strains and to identify the gene loci involved in appearance of a color spot by microscopical pigment analysis (Fahrig, 1984; Fahrig and Neuh~iuserKlaus, 1985). The embryos treated have to be the F 1 from the cross C57B1 × T, being homozygous for nonagouti ( a / a ) , and heterozygous for brown ( b / B ) , pink-eyed dilution and chinchilla (p ceh/ P C), dilute and short ear (d s e / D SE), and piebald spotting (s/S). Mutations of piebald spotting or short ear cannot be detected using the spot test. As can be seen in Fig. 3, heterozygosity of the recessive mutant alleles leads to a dark gray coat in the F I. In contrast to this the mother animal, being homozygous for the wild-type alleles, has a black coat. Gene mutations can now be detected as genetic alterations at the c locus; cCh/c ch in combination with a / a results in a dull black or sepia color spot neither of which contrasts clearly with the coat. However, the genetic alteration that can be detected is a mutation of c, or a lethal 'allele' of c, both of which combined with c ch give rise to
a light brown c / c ch phenotype. Therefore, light brown spots are caused only by gene mutation or small deletions, but not by recombinations (Fig.
3). Beyond that it is possible to detect reciprocal recombinations between the p and c loci because the loci are located on the same chromosome (14 units apart). A genetic alteration leading to p c c h / p Cch or p cch/A (A = deletion) gives rise to near-white color spots, the characteristic reduc-
no g e n e t i c a l t e r a t i o n :
heterozygous
ch
p
c
p
c
°'°~°
ch
•4-
"4-
-I-
.4no spot
dark tray coat
g e n e m u t a t i o n + to c :
heterozYsous mutant alleles ch
p
|fEb! browll Spe!
c
2 ~ i i : ~ m ¢h
Fig. 3. Diagrammatic representation of how to distinguish between induced gene mutation and induced reciprocal recombination due to mitotic crossing-over. Chromosome 7 of the mouse, heterozygous at the c and p loci. The homologue chromosomes are duplicated structures consisting of two identical sister chromatids. Mitosis leads to two daughter cells with the parental heterozygous genotype, i.e., will lead to a coat with dark gray color. A mutation of the wild-type allele at the c locus, ( + ~ c) leads in one of the two daughter cells to heterozygosity of the mutant alleles (c/cob), giving rise to light brown spots. cCh/cCh and + / c eh would result in a dark gray color, light brown spots can only be the result of a gene mutation and not of a recombination event or chromosome loss. A crossing-over between centromere and the two gene loci under study leads to an exchange of the mutant and the wild-type alleles. Mitosis leads to homozygosity of the wild-type alleles in one of the daughter cells and to homozygosity of the mutant alleles in the other one. Homozygous wild-type alleles can be seen as black spots on the dark gray coat, homozygous m u t a n t alleles as near-white spots. If both reciprocal products survive, a twin spot may arise. No genetic alteration other than reciprocal recombination will result in twin spots.
As
P
C
4-
C
4 ~"ili:-'~~
lira
÷
-4no s p o t
dark
gray
reciprocal recombination : h o m o z y g o u s w i l d type b l a c k spot
2 ~:
+
~ .
.
.
.
.
homozygous mutant alleles near-white spot
182
tion in pigmentation being clearly identifiable by microscopical analysis. Near-white spots are unlikely to be due to gene mutations, since simultaneous mutations at the linked loci p and c are extremely rare and have never been observed in specific-locus experiments. It is also highly unlikely that large deletions involving both the c and p loci are sufficiently viable in the heterozygous form or in the case of monosomy. The most likely genetic alteration leading to viable ceils of the genotype p Cch/p Cch is reciprocal recombination due to mitotic crossing-over. The corresponding reciprocal products of mitotic crossing-over are cells of the genotype P C / P C. The detection of P C / P C is possible since the recessive genes, even in the heterozygous state, have an influence on the level of pigmentation. In contrast to the homozygous nonagouti black mother animals, F~ animals are dark gray to black on the back, and medium gray on the ventral side. Therefore, pigment cells of the genotype P C / P C show up as black spots (Fig. 3). Color pictures of spots and hair pigment have been published recently (Fahrig and Neuh~iuser-Klaus, 1985). A feature of mitotic crossing-over is the potential for forming twin spots. As can be seen in Fig. 3, twin spots, homozygous for the recessive markers and their wild-type alleles respectively, are both distinguishable from the heterozygous remainder of the body. It is not necessary that both spots should be visible; the descendants of either
of the daughter ceils may not occupy a position on the surface, or where the marker gene can express itself. Therefore, the appearance of twin spots is a rare event. Table 2 shows that recombinations themselves are no rare events, even though twin spots rarely occur. The strong direct-acting alkylating agent ethyl nitrosourea (ENU), known as the most potent inducer of gene mutations in mammals (Russell et al., 1979), induced in a concentration of 30 m g / k g about 14% F~ animals with color spots of genetic relevance (SGR). Of the color spots induced about 13% were light brown, i.e., products of gene mutations, and about 8% were near-white, black, or black and near-white twin spots. As can be seen in Fig. 3, only 50% of all reciprocal recombinations will lead to color spots while all gene mutations of + ~ c will lead to a light brown color spot. Therefore, the frequency of gene mutations and reciprocal recombinations may not be too far different. The same tendency is visible within the control. As concerns the gray and brown color spots it is not possible to distinguish between the different genetic alterations which may be involved in their appearance. As can be seen in Table 2, the frequency of twin spots induced by ENU was only 0.2%. As ENU is known to be a potent inducer of gene mutations (Russell et al., 1979) it may well be that there exist substances inducing preferentially recombinations. A certain trend in this direction
TABLE 2 D I S T R I B U T I O N OF C O L O R SPOTS A M O N G F O U R G E N E LOCI IN T H E M A M M A L I A N SPOT T E S T Original state Color of spot 858 color spots ~ induced with E N U and identified by microscopical analysis were 39 color spots 2 induced with caprolactam and identified by microscopical analysis 27 color spots 3 within the control identified by microscopical analysis were
b/B Brown 132 (15%) 2 (5%) 5 (19%)
p/P Light gray 259 (30%) 12 (31%) 6 (22%)
d/D Gray 281 (33%) 18 (46%) 10 (37%)
c~h/C Light brown 115 (13%) 0 3 (11%)
p cCh/P C Near-white 53 (6%) 4 (10%) 3 (11%)
Twin spot 2 (0.2%) 1 (2.5~) 0
Maternal black 16 (2%) 2 (5%) 0
Pooled experiments 1981-1990; treatment: 30 m g / k g ethylnitrosourea (ENU); treated females with litter: 1044; F t checked for color spots: 5887; F l with spots of genetic relevance (SGR): 796 = 13.5% (58 animals with 2 spots of different color, 2 animals with 3 spots of different color). 2 Pooled experiments; treatment: 400 or 500 m g / k g caprolactam; treated females with litter: 282; F 1 checked for color spots: 1 781; F~ with SGR: 39 = 2.2%. Pooled control data; treatment: 0.1 ml solvent or 0.01 ml D M S O per 10 g body weight; females with litter: 472; F I checked for color spots: 2796: F t with SGR: 26 = 0.93% (1 animal with 2 SGR).
183
may perhaps be shown by caprolactam (Fahrig, 1989). This substance induces chromosomal aberrations in cultured cells and is weakly active as both a somatic cell and a germ-cell mutagen in female Drosophila (review: Ashby and Shelby, 1989). Caprolactam was only weakly active in the spot test. But of the color spots induced, 17.5% were the presumed products of reciprocal recombinations, and the frequency of twin spots was 2.5%. Light brown spots could not be observed at all. The most promising wa~ of enhancing recombinagenic effects is to combine a mutagen with a tumor promoter. The corresponding experiments are described in another paper of this special issue of Mutation Research. References Ashby, J. and M.D. Shelby (1989) Overview of the genetic toxicity of caprolactam and benzoin, Mutation Res., 224, 321-324. Bateman, A.J. (1967) A probable case of mitotic crossing-over in the mouse, Genet. Res. Camb., 9, 375-376. Carter, T.C. (1952) A mosaic mouse with an anomalous segregation ratio, J. Genet., 51, 1-6. Castle, W.E. (1922) On a non-transmissible tri-color variation in rats, Carnegie Inst. Wash. Publ. No. 320, pp. 51-54. Curtis, M.R., and W.F. Dunning (1940) An independent recurrence of the blue mutation in the Norway rat and a blue-black mosaic, J. Hered., 31,219-222. Fahrig, R. (1975) A mammalian spot test: induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens, Mol. Gen. Genet., 138, 309-314. Fahrig, R. (1976) The effect of dose and time on the induction of genetic alterations in Saecharomyces cerevisiae by aminoacridines in the presence and absence of visible light irradiation in comparison with the dose-effect curves of mutagens with other types of action, Mol. Gen. Genet., 144, 131-140. Fahrig, R. (1984) Genetic mode of action of cocarcinogens and tumor promoters in yeast and mice, Mol. Gen. Genet., 194, 7-14. Fahrig, R. (1989) Possible recombinogenic effect of Caprolactam in the mammalian spot test, Mutation Res., 224, 373-375. Fahrig, R., and A. Neuh~iuser-Klaus (1985) Similar pigmenta-
tion characteristics in the specific locus and the mammalian spot test: a way to distinguish between induced mutation and recombination, J. Hered., 76, 421-426. Feldman, H.W. (1935) A mosaic (dark-eyed intense-pink-eyed dilute) coat colour of the house mouse, J. Genet., 30, 383-388. Fisher, G., D.A. Stephenson and J.D. West (1986) Investigation of the potential for mitotic recombination in the mouse, Mutation Res., 164, 381-388. Fisher, R.A. (1929) Note on a tricolour (mosaic) mouse, J. Genet., 23, 77-81. Griineberg, H. (1966) The case for somatic crossing-over in the mouse, Genet. Res., 7, 58-75. Hastings, P.J. (1988) Recombination in the eukaryotic nucleus, BioEssays, 9, 61-64. Keeler, C.E. (1931) A probable new mutation to white-belly in the house mouse, Mus musculus, Proc. Natl. Acad. Sci. (U.S.A.), 17, 700-703. Liskay, R.M., and J.L. Stachelek (1983) Evidence for intrachromosomal gene conversion in cultured mouse cells, Cell, 35, 157-165. Morgan, W.C., and S.P. Holman (1955) Eight mosaic mice in thirty years, J. Hered., 46, 47-48. Pickard, J.N. (1936) A black-blue Dutch rabbit, J. Genet., 33, 337-341. Pincus, G. (1929) A mosaic (black-brown) coat pattern in the mouse, J. Exp. Zool., 52, 439-441. Russell, L.B. (1971) Definition of functional units in a small chromosomal sequence of the mouse and its use in interpreting the nature of radiation induced mutations, Mutation Res., 11, 107-123. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, W.L., E.M. Kelly, P.R. Hunsicker, J.W. Bangham, S.C. Maddux and E.I. Phipps (1979) Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse, Proc. Natl. Acad. Sci. (U.S.A.), 76, 5818-5819. Seperack, P.K., M.C. Strobel, DJ. Corrow, N.A. Jenkins and N.G. Copeland (1988) Somatic and germ-line reverse mutation rates of the retrovirus-induced dilute coat color mutation of DBA mice, Proc. Natl. Acad. Sci. (U.S.A.) 85, 189-192. Silvers, W.K. (1979) Dominant-spotting. Patch, and rumpwhite, in: The Coat Colors of Mice: A Model for Gene Action and Interaction, Springer, New York, pp. 206-241. Stern, C. (1936) Somatic crossing-over and segregation in Drosophila melanogaster, Genetics, 21,625-730. Wright, S., and O.N. Eaton (1926) Mutational mosaic coat patterns of the guinea pig, Genetics, 11,333-351.
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 00383
Tests for recombinagens in mammals in vivo Rudolf Fahrig Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Abteilung Genetik, Hannover, Germany (Accepted 30 March 1992)
Keywords: Reciprocal recombination; Heterozygous recessive mutations; Spot test
History In rare instances heterozygous animals show a patch of fur for the recessive phenotype, and then the gene has definitely affected the coat. Recombinational processes rather than gene mutations belong to the most potent mechanisms for recessive genes to come to expression. Justification for this statement comes from experiments using yeast, in which the probability that a heterozygous recessive gene becomes homozygous is two orders of magnitude higher for nonreciprocal recombination than for gene mutation (Fahrig, 1976). Also, observations in cultured mouse cells showed that the frequency of nonreciprocal recombination (gene conversion) between repetitive genes is several orders of magnitude higher than the frequency of gene mutation (Liskay and Stachelek, 1983). With reciprocal recombination a single event is sufficient to result in the expression of all recessive mutations of a chromosomal segment, whereas with gene mutations several single events would be needed in order to achieve a similar effect. In spite of that, as possible mechanisms in mammals they have not often been considered. The reason for this is that mitotic chromosome pairing has not been observed in mammals.
Correspondence: Dr. R. Fahrig, Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Abteilung Genetik, Nikolai-Fuchs-Str. 1, D-3000 Hannover 61, Germany.
Nevertheless, 5 years before Stern's (1936) classical work with Drosophila, Keeler (1931) considered the possibility of mitotic crossing-over, i.e., reciprocal recombination in mice. But shying away from this idea, he proceeded to give a different interpretation of his data. Therefore, the first case with convincing evidence for mitotic reciprocal recombination in mammals as mechanism excluding somatic mutation as a possible cause was (Carter, 1952) a mosaic mouse born in 1948 in the first of her parents' three litters. Her father was of the wild-type CBA inbred strain; her mother was a cross-bred heterozygote for the semidominant color and macrocytic anemia mutant W v. The mosaic showed, over the greater part of her body, the characteristic phenotype of a W V / + heterozygote; there was a white brown spot, a large white belly patch, the agouti hairs of the dorsum were bleached and the ventral surface was silvered. Two regions, however, showed full, wild-type coloration. The greater part of the wild-type coloration was on the right side of the mouse, and this position suggested that the right gonad, or possibly both, might have been involved; the mosaic was therefore mated to a wild-type male to test the segregation of W v. Of the 41classified young, 10 were wild-type and 31 were typical W V / + heterozygotes; the deviation from the expected 1 : 1 ratio was significant statistically (P = 0.00145). None showed any sign of mosaicism. The data established that the mosaic female developed from a W V / + zygote, that parts of her
178
soma were deficient of W v, and that she bred as though her germinal tissues were partly deficient of +w. Somatic mutation could hardly have been the mechanism of formation, since two mutational steps would have to be postulated, W v to + in the soma and + to W v in the gonad. Deletion likewise fails to provide an explanation, since two deletions, in homologous chromosomes, would have to be postulated. The only possible explanation is given by mitotic recombination. The W" mosaic mouse would be supposed to have arisen by crossing-over proximal to the locus of W v, at an early cleavage division. The daughter cells would have the constitutions W v / w v and + / + and would have given rise to one ovary and to the fully colored patches respectively. The affected ovary would produce only WV-bearing ova; the unaffected ovary would produce W v- and +-bearing ova in equal numbers; and the expected segregation ratio would again be 3 : 1, agreeing well with observation (Fig. 1). Grfineberg (1966) extracted material from the literature to show that mosaicism in mammals is best explicable by mitotic crossing-over and not by mutation. He discussed nearly all published cases of mosaicism in mice (Pincus, 1929; Fisher, 1931; Feldman, 1935; Morgan and Holman, 1955; Russell and Major, 1957), rats (Castle, 1922; Curtis and Dunning, 1940), rabbits (Castle, 1922; Pickard, 1936) and guinea pigs (Wright and Eaton, 1926). But in conclusion he had to admit that also other mechanisms than recombination due to crossing-over have to be considered for expres-
Gonads
Soma
Fig. 1. Interpretation of Carter's case in terms of mitotic crossing-over.
b !Oiiii~i~iiiiii!ilililililiiii!
deletion or • reciprocal b
chromosome loss
I
b
b
+
b
recombination
I
nonreciprocal ~r
b
gene mutation
recombination b
b Fig. 2. Different ways for a heterozygous recessive mutation to come to expression.
sion of the recessive genes. Fig. 2 shows that other possible mechanisms are gene mutation, chromosome loss, non-disjunction, chromosome deletion, or nonreciprocal recombination due to gene conversion * As an addendum to Grfineberg's extensive survey of evidence for mitotic crossing-over in mammals (1966), Bateman (1967) published a short note. In contrast to the case of Carter (1952), the case which was the subject of this note was un-
There are two distinct possible genetic consequences of a recombinational interaction. They are known as reciprocal recombination due to crossing-over and non reciprocal recombination due to gene conversion. Crossing-over is seen as a reciprocal breakage and rejoining of homologous molecules such that all information on one side of the crossing-over is recombined reciprocally with all information on the other side. Mitotic crossing-over is detected as homozygosis for all markers distal to the crossing-over. Mitotic crossing-over can lead to homozygosis only when it occurs in G2 (the four strand stage). Mitotic gene conversion can occur in GI and is defined as a nonreciprocal transfer of information. This type of mitotic recombination is a repair process which is known to repair double strand breaks and to fill double strand gaps by copying a homologous sequence. The words crossing-over and conversion are used to denote not only the genetic outcome of these recombinational processes but also the processes themselves. (Hastings, 1988)
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doubtedly somatic in origin. Mitotic crossing-over differs from all other phenomena which may have an outward resemblance to it, by producing two complementary genotypes, though one of these is usually indistinguishable phenotypically from the heterozygous state from which it arose. In the case described by Bateman (1967) both homozygotes were distinguishable from the heterozygote. The mouse was non-agouti and heterozygous for c ch (chinchilla) and c (albino). The heterozygote is fawn-colored (light brown) and the homozygotes are dark gray, also called sepia (cch/cch), and white (c/c). Somatic crossing-over between the c-locus and the centromere had produced just such a twin-spotting of dark gray and white as could be expected theoretically. Present
Fisher et al. (1986) have made an attempt to distinguish between reciprocal recombination and somatic mutation or non-disjunction in mice. But they found little evidence for mitotic recombination following X-irradiation of 10.25-day-old embryos heterozygous in repulsion for pink-eyed dilution (p) and albino (c), two linked loci assigned to chromosome 7. The reason for not having succeeded was that for lack of microscopical hair analysis, the authors could not address the question as to whether the presence of white spots was due to genetic alterations expressing the albino genotype or to pigment cell death. As nearly all spots observed by them were white, an interpretation of results was not possible at all. A more convincing report about spontaneous and induced mitotic recombination comes from Panthier et al. (1990). In this report, phenotypic reversions of the W ei allele at the W locus were studied. Mice heterozygous in repulsion for both W ei and buff (bf) (i.e., W ei + / + bf) were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khakicolored coat of nonagouti buff homozygotes ( a / a : bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a / a : W ei + / + b f mice: either solid black or khaki-colored.
Furthermore phenotypic reversions o f w e i / q were enhanced significantly following X-irradiation of 9.25-day-old W e i / + embryos ( P < 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. Twin spots with w e i + / w e i + and + b f / + bf genotypes respectively would have made a much stronger case for mitotic recombination. No such twin spots were observed by Panthier et al. (1990). However, they were probably not observable because all available evidence indicates that death of melanoblasts is responsible for the white spotting of the coats of W locus mutants (Silvers, 1979). Accordingly; a W ei q- / W ei q- homozygous clone would be expected to die, leaving an area which might be colonized or not by W e~ + / + bf or + b f / + bf surrounding melanoblasts. Similarly a single mitotic crossing-over between the W locus and the bf locus would be expected to produce a clone of w e i b f / + bf melanocytes. Again no such clones were observed. This presumably results from the inability to detect w e i b f / + bf patches, composed of white hairs interspersed among khaki rather than black hairs, on the coat of W ei + / + bf mice. It has recently become possible to detect mitotic reciprocal recombination at the dilute locus of mice in vivo by using methods of molecular biology. The dilute locus has served as a useful visual marker in a number of studies designed to measure mutation rates in the mouse following X-ray or chemical treatment. For example, in a study of Russell (1971), 235 radiation-induced mutations involving the dilute locus and a closely linked marker, short ears (within 0.1 cM), were identified. Complementation tests revealed 16 different complementation groups. Four classes of forward mutations to dilute were identified including dilute, viable ('d'); dark dilute, viable (d*); dilute, opisthotonic (d°P); and prenatally lethal (dPJ). As these mutations are X-ray-induced, many of them may represent deletions or DNA rearrangements. In some cases, dilute mutations were identified that included both dilute and short ears and thus probably represent large deletions. At present, the dilute (d v) coat color mutation of DBA mice provides the only simple means for
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measuring the relative somatic and germ-line reverse mutation rates of retrovirus-induced mutations in mammals. The d v mutation was generated by the spontaneous integration of an ecotropic murine leukemia virus into noncoding sequences of the dilute locus. Reversion of the d v mutation occurs by provirus excision and is mediated by homologous recombination events involving the viral long terminal repeat sequences. Although numerous independent germ-line d + revertants have been identified, only one case of somatic d + rcvertants has been reported. Over 5 years, Seperack et al. (1988) have screened more than one million mice homozygous for the d v mutation to determine whether they could identify somatic d v revertants. This survey has resulted in the identification of one somatic d + revertant. By Southern blot analysis of EcoRI-digested DNA from the somatic revertant, the authors were able to show that the revertant examined appeared to be the result of reciprocal recombination between homologous chromosomes. Test system Instead of checking one million mice for spontaneously occurring color spots, it is more sensible to induce such spots by treatment with chemical mutagens or even X-rays, which were used by Russell and Major (1957) in their pioneer experiments. The only suitable test system to detect reciprocal recombinations is the spot test with mice (Fahrig, 1975). According to this method mouse embryos, which are heterozygous for different recessive coat color genes, are treated in utero
between the 9th and 1 lth day post conception by injection of a mutagen into the peritoneal cavity of the mother animal, or by other appropriate routes of administration. If this treatment leads to an alteration or loss of a specific wild-type allele in a pigment precursor cell, a color spot in the coat of the adult animal may appear. With regard to the mechanism (see Fig. 2) by which the heterozygous recessive coat color alleles can be expressed, this is either a gene mutation, theoretically also loss of the wild-type allele through deletion or monosomy, or a recombinational process such as mitotic crossing-over (reciprocal recombination), or mitotic gene conversion (nonreciprocal recombination). Of the numerical and structural chromosome aberrations that can lead to loss of the wild-type allele, only those that survive several mitoses would cause a spot with expression of the recessive allele. In the course of the routinely performed spot test, three types of spots are distinguished: (1) white midventral spots (which have no pigment at all), regarded as resulting from pigment cell killing; (2) spots with hairs similar to the yellow hairs which normally surround ears, genital papillae, and mammae, classified as misdifferentiation spots and appearing as yellow fluorescent hairs with agouti genotype; (3) spots of genetic relevance (SGR) resulting from genetic alterations at the different gene loci (Table 1) and expressing the recessive mutant or their wild-type alleles. It is apparent that without routinely performed microscopical analysis it is not possible to distinguish between the different mechanisms leading to expression of a recessive mutation. Therefore, nearly all published work about the spot test cannot be used for the specific subject of this
TABLE 1 THEORETICAL EXPECTIONS Original state
b/B
Theoretically color spots can be induced by Mutation (a) b,/b ~ Deletion ( J ) b/zl Monosomy (0) b/0 Reciprocal recombination b/b Nonreciprocal recombination (*) b/b* Color of spot Brown
p/P
d/D
cch/c
p/p~ p/3 p/p p/p* Light gray
d/da d/A d/0 d/d d/d* Gray
cCh/c a cCh/A Light brown
p cCh/p C
(p cCh/A) (p cch/0) -p c c h / p c ch twin spot
(P C / A ) (P C / 0 ) PC/PC
Near-white
Maternal black
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review. The only possibility to distinguish between induced mutations and induced reciprocal recombinations is to use specific mouse strains and to identify the gene loci involved in appearance of a color spot by microscopical pigment analysis (Fahrig, 1984; Fahrig and Neuh~iuserKlaus, 1985). The embryos treated have to be the F 1 from the cross C57B1 × T, being homozygous for nonagouti ( a / a ) , and heterozygous for brown ( b / B ) , pink-eyed dilution and chinchilla (p ceh/ P C), dilute and short ear (d s e / D SE), and piebald spotting (s/S). Mutations of piebald spotting or short ear cannot be detected using the spot test. As can be seen in Fig. 3, heterozygosity of the recessive mutant alleles leads to a dark gray coat in the F I. In contrast to this the mother animal, being homozygous for the wild-type alleles, has a black coat. Gene mutations can now be detected as genetic alterations at the c locus; cCh/c ch in combination with a / a results in a dull black or sepia color spot neither of which contrasts clearly with the coat. However, the genetic alteration that can be detected is a mutation of c, or a lethal 'allele' of c, both of which combined with c ch give rise to
a light brown c / c ch phenotype. Therefore, light brown spots are caused only by gene mutation or small deletions, but not by recombinations (Fig.
3). Beyond that it is possible to detect reciprocal recombinations between the p and c loci because the loci are located on the same chromosome (14 units apart). A genetic alteration leading to p c c h / p Cch or p cch/A (A = deletion) gives rise to near-white color spots, the characteristic reduc-
no g e n e t i c a l t e r a t i o n :
heterozygous
ch
p
c
p
c
°'°~°
ch
•4-
"4-
-I-
.4no spot
dark tray coat
g e n e m u t a t i o n + to c :
heterozYsous mutant alleles ch
p
|fEb! browll Spe!
c
2 ~ i i : ~ m ¢h
Fig. 3. Diagrammatic representation of how to distinguish between induced gene mutation and induced reciprocal recombination due to mitotic crossing-over. Chromosome 7 of the mouse, heterozygous at the c and p loci. The homologue chromosomes are duplicated structures consisting of two identical sister chromatids. Mitosis leads to two daughter cells with the parental heterozygous genotype, i.e., will lead to a coat with dark gray color. A mutation of the wild-type allele at the c locus, ( + ~ c) leads in one of the two daughter cells to heterozygosity of the mutant alleles (c/cob), giving rise to light brown spots. cCh/cCh and + / c eh would result in a dark gray color, light brown spots can only be the result of a gene mutation and not of a recombination event or chromosome loss. A crossing-over between centromere and the two gene loci under study leads to an exchange of the mutant and the wild-type alleles. Mitosis leads to homozygosity of the wild-type alleles in one of the daughter cells and to homozygosity of the mutant alleles in the other one. Homozygous wild-type alleles can be seen as black spots on the dark gray coat, homozygous m u t a n t alleles as near-white spots. If both reciprocal products survive, a twin spot may arise. No genetic alteration other than reciprocal recombination will result in twin spots.
As
P
C
4-
C
4 ~"ili:-'~~
lira
÷
-4no s p o t
dark
gray
reciprocal recombination : h o m o z y g o u s w i l d type b l a c k spot
2 ~:
+
~ .
.
.
.
.
homozygous mutant alleles near-white spot
182
tion in pigmentation being clearly identifiable by microscopical analysis. Near-white spots are unlikely to be due to gene mutations, since simultaneous mutations at the linked loci p and c are extremely rare and have never been observed in specific-locus experiments. It is also highly unlikely that large deletions involving both the c and p loci are sufficiently viable in the heterozygous form or in the case of monosomy. The most likely genetic alteration leading to viable ceils of the genotype p Cch/p Cch is reciprocal recombination due to mitotic crossing-over. The corresponding reciprocal products of mitotic crossing-over are cells of the genotype P C / P C. The detection of P C / P C is possible since the recessive genes, even in the heterozygous state, have an influence on the level of pigmentation. In contrast to the homozygous nonagouti black mother animals, F~ animals are dark gray to black on the back, and medium gray on the ventral side. Therefore, pigment cells of the genotype P C / P C show up as black spots (Fig. 3). Color pictures of spots and hair pigment have been published recently (Fahrig and Neuh~iuser-Klaus, 1985). A feature of mitotic crossing-over is the potential for forming twin spots. As can be seen in Fig. 3, twin spots, homozygous for the recessive markers and their wild-type alleles respectively, are both distinguishable from the heterozygous remainder of the body. It is not necessary that both spots should be visible; the descendants of either
of the daughter ceils may not occupy a position on the surface, or where the marker gene can express itself. Therefore, the appearance of twin spots is a rare event. Table 2 shows that recombinations themselves are no rare events, even though twin spots rarely occur. The strong direct-acting alkylating agent ethyl nitrosourea (ENU), known as the most potent inducer of gene mutations in mammals (Russell et al., 1979), induced in a concentration of 30 m g / k g about 14% F~ animals with color spots of genetic relevance (SGR). Of the color spots induced about 13% were light brown, i.e., products of gene mutations, and about 8% were near-white, black, or black and near-white twin spots. As can be seen in Fig. 3, only 50% of all reciprocal recombinations will lead to color spots while all gene mutations of + ~ c will lead to a light brown color spot. Therefore, the frequency of gene mutations and reciprocal recombinations may not be too far different. The same tendency is visible within the control. As concerns the gray and brown color spots it is not possible to distinguish between the different genetic alterations which may be involved in their appearance. As can be seen in Table 2, the frequency of twin spots induced by ENU was only 0.2%. As ENU is known to be a potent inducer of gene mutations (Russell et al., 1979) it may well be that there exist substances inducing preferentially recombinations. A certain trend in this direction
TABLE 2 D I S T R I B U T I O N OF C O L O R SPOTS A M O N G F O U R G E N E LOCI IN T H E M A M M A L I A N SPOT T E S T Original state Color of spot 858 color spots ~ induced with E N U and identified by microscopical analysis were 39 color spots 2 induced with caprolactam and identified by microscopical analysis 27 color spots 3 within the control identified by microscopical analysis were
b/B Brown 132 (15%) 2 (5%) 5 (19%)
p/P Light gray 259 (30%) 12 (31%) 6 (22%)
d/D Gray 281 (33%) 18 (46%) 10 (37%)
c~h/C Light brown 115 (13%) 0 3 (11%)
p cCh/P C Near-white 53 (6%) 4 (10%) 3 (11%)
Twin spot 2 (0.2%) 1 (2.5~) 0
Maternal black 16 (2%) 2 (5%) 0
Pooled experiments 1981-1990; treatment: 30 m g / k g ethylnitrosourea (ENU); treated females with litter: 1044; F t checked for color spots: 5887; F l with spots of genetic relevance (SGR): 796 = 13.5% (58 animals with 2 spots of different color, 2 animals with 3 spots of different color). 2 Pooled experiments; treatment: 400 or 500 m g / k g caprolactam; treated females with litter: 282; F 1 checked for color spots: 1 781; F~ with SGR: 39 = 2.2%. Pooled control data; treatment: 0.1 ml solvent or 0.01 ml D M S O per 10 g body weight; females with litter: 472; F I checked for color spots: 2796: F t with SGR: 26 = 0.93% (1 animal with 2 SGR).
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may perhaps be shown by caprolactam (Fahrig, 1989). This substance induces chromosomal aberrations in cultured cells and is weakly active as both a somatic cell and a germ-cell mutagen in female Drosophila (review: Ashby and Shelby, 1989). Caprolactam was only weakly active in the spot test. But of the color spots induced, 17.5% were the presumed products of reciprocal recombinations, and the frequency of twin spots was 2.5%. Light brown spots could not be observed at all. The most promising wa~ of enhancing recombinagenic effects is to combine a mutagen with a tumor promoter. The corresponding experiments are described in another paper of this special issue of Mutation Research. References Ashby, J. and M.D. Shelby (1989) Overview of the genetic toxicity of caprolactam and benzoin, Mutation Res., 224, 321-324. Bateman, A.J. (1967) A probable case of mitotic crossing-over in the mouse, Genet. Res. Camb., 9, 375-376. Carter, T.C. (1952) A mosaic mouse with an anomalous segregation ratio, J. Genet., 51, 1-6. Castle, W.E. (1922) On a non-transmissible tri-color variation in rats, Carnegie Inst. Wash. Publ. No. 320, pp. 51-54. Curtis, M.R., and W.F. Dunning (1940) An independent recurrence of the blue mutation in the Norway rat and a blue-black mosaic, J. Hered., 31,219-222. Fahrig, R. (1975) A mammalian spot test: induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens, Mol. Gen. Genet., 138, 309-314. Fahrig, R. (1976) The effect of dose and time on the induction of genetic alterations in Saecharomyces cerevisiae by aminoacridines in the presence and absence of visible light irradiation in comparison with the dose-effect curves of mutagens with other types of action, Mol. Gen. Genet., 144, 131-140. Fahrig, R. (1984) Genetic mode of action of cocarcinogens and tumor promoters in yeast and mice, Mol. Gen. Genet., 194, 7-14. Fahrig, R. (1989) Possible recombinogenic effect of Caprolactam in the mammalian spot test, Mutation Res., 224, 373-375. Fahrig, R., and A. Neuh~iuser-Klaus (1985) Similar pigmenta-
tion characteristics in the specific locus and the mammalian spot test: a way to distinguish between induced mutation and recombination, J. Hered., 76, 421-426. Feldman, H.W. (1935) A mosaic (dark-eyed intense-pink-eyed dilute) coat colour of the house mouse, J. Genet., 30, 383-388. Fisher, G., D.A. Stephenson and J.D. West (1986) Investigation of the potential for mitotic recombination in the mouse, Mutation Res., 164, 381-388. Fisher, R.A. (1929) Note on a tricolour (mosaic) mouse, J. Genet., 23, 77-81. Griineberg, H. (1966) The case for somatic crossing-over in the mouse, Genet. Res., 7, 58-75. Hastings, P.J. (1988) Recombination in the eukaryotic nucleus, BioEssays, 9, 61-64. Keeler, C.E. (1931) A probable new mutation to white-belly in the house mouse, Mus musculus, Proc. Natl. Acad. Sci. (U.S.A.), 17, 700-703. Liskay, R.M., and J.L. Stachelek (1983) Evidence for intrachromosomal gene conversion in cultured mouse cells, Cell, 35, 157-165. Morgan, W.C., and S.P. Holman (1955) Eight mosaic mice in thirty years, J. Hered., 46, 47-48. Pickard, J.N. (1936) A black-blue Dutch rabbit, J. Genet., 33, 337-341. Pincus, G. (1929) A mosaic (black-brown) coat pattern in the mouse, J. Exp. Zool., 52, 439-441. Russell, L.B. (1971) Definition of functional units in a small chromosomal sequence of the mouse and its use in interpreting the nature of radiation induced mutations, Mutation Res., 11, 107-123. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, W.L., E.M. Kelly, P.R. Hunsicker, J.W. Bangham, S.C. Maddux and E.I. Phipps (1979) Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse, Proc. Natl. Acad. Sci. (U.S.A.), 76, 5818-5819. Seperack, P.K., M.C. Strobel, DJ. Corrow, N.A. Jenkins and N.G. Copeland (1988) Somatic and germ-line reverse mutation rates of the retrovirus-induced dilute coat color mutation of DBA mice, Proc. Natl. Acad. Sci. (U.S.A.) 85, 189-192. Silvers, W.K. (1979) Dominant-spotting. Patch, and rumpwhite, in: The Coat Colors of Mice: A Model for Gene Action and Interaction, Springer, New York, pp. 206-241. Stern, C. (1936) Somatic crossing-over and segregation in Drosophila melanogaster, Genetics, 21,625-730. Wright, S., and O.N. Eaton (1926) Mutational mosaic coat patterns of the guinea pig, Genetics, 11,333-351.