ANALYSIS OF THE ALBINO-LOCUS REGION O F THE MOUSE.
11. MOSAIC MUTANTS*
LIANE
B. RUSSELL
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Manuscript received December 30, 1977
Revised copy received July 25, 1978
ABSTRACT
Among 119 mutations involving the c locus that were recovered in the
course of mouse specific-locus experiments with external radiations, 16 were
found in mosaic, or fractional, mutants. The number of additional c-locus
fractionals that could have occurred i n these experiments and, for a variety
of reasons, might not have been clearly identified, probably does not exceed
the present number.There was no evidence for radiation induction of
the fractionals, and even those cccurring in the irradiated groups may thus be
assumed to be of spontaneous origin. Since only two mutations in the control
groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mu tations are fractionahNone of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except
for one subvital and one not tested.Genetic tests of the fractionals
indicated no major selection against the new mutations, either gametically or
For the group of fractionals as a whole, about one-half of
i n the progeny.the germinal tissue carried the mutation, indicating that the fractionals came
from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a
daughter chromosome derived from pronuclear DNA synthesis of the zygote, or
in one of the first two blastomeres prior to replication. Since the mouse embryo
does not stem from all of the cleavage products of the zygote, the frequency
of fractionals observed underestimates the frequency of mutational events that
result in two types of blastomeres.
UTATIONS involving the c-locus recovered in the course of a large number
of specific-locus experiments on mice have recently been analyzed with
respect to their origin and viability (L. B. RUSSELL,W. L. RUSSELLand KELLY
1979; RUSSELLand RAYMER1979). Among a total of 119 c-locus mutants recovered in experiments designed to test the mutagenic effects of externally administered radiations of various types, there were 16 mosaic, or fractional, mutants.
These fractionals have special significance with regard to conclusions that may
be drawn concerning both the time of occurrence of spontaneous mutations and
the mutational spectrum observed following various radiation treatments. For
these reasons, this subset of c-locus mutants is examined by itself in this paper.
* Research sponsored by the Division of Biomedical and Environmental Research, U. S . Department
Contract W-7405-eng-26with the Union Carbide Corporation.
Genetics 91 : 141-147 January, 1979.
of Energy under
142
L. B. RUSSELL
MATERIALS AND METHODS
As was the case for c-locus whole-body mutants, fractionals (with one exception) were recovered i n crosses of genetically uniform homozygous wild-type mice to a noninbred multiplerecessive stock which, with regard to the c-locus, was cch/cch. The stocks used, the full genotypes
and phenotypes involved, and the testing procedure are described in the first paper of this series
(RUSSELL, RUSSELLand KELLY 1979). Fractional mutants were recognized by area(s) of lighter
fur, or by mottling, except for one c-locus mosaic discovered among the wild-type parents of the
usual cross by virtue of its progeny (symbol Cr in the tables).
Since all fractionals, with the exception of this one, derive from C/C x cch/cch crosses, one
of the genotypes making up each mosaic is C/cch. The other genotype is c*/cCh, where the
general designation C* is used for the mutant allele a t the c locus. Therefore, 15 of the 16 fractionals were of the type c*/cch///C/cch, and one (Cr) was c*/C///C/C. Subclasses of c* are
designated on the basis of their phenotype and viability (RUSSELL,RUSSELLand KELLY 1979).
When mottled probands are found in specific-locus experiments, it is often very difficult to
identify the phenotype of the nonwild-type portions of the fur. Mottleds were, therefore, usually
test mated for several of the specific loci. Three types of c-locus mosaics might have escaped
definitive identification (a) those which produced insufficient progeny from cch/cch or c/c
mates to determine the existence and (if so) the magnitude of germinal involvement; (b) probands in which the mutation was to cch, so that germinal involvement, if it existed, could only
rather than from the transmission
be surmised from progeny ratios (i.e.,a possible excess of
of a n allele not present i n the parents; (c) a very few probands mottled with near-white that
could have been mosaic for long deletions, including both c and p loci and not viable in progeny.
Earlier tabulations made from a subset of specific-locus experiments (RUSSELL1964.) indicate
that any c-locus fractionals that might have been among the group of mottleds not identified
as to the locus at which they were mosaic probably did not exceed in frequency the number
of clearly identified c-locus mosaics.
It is also possible that probands i n which only a very small portion of the fur was mosaic
might have missed detection. Our experience with somatic mutation experiments, however,
indicates that mutant areas smaller than 0.01% of the body surface are detectable on a n a/a
background (RUSSELLand MAJOR1957), and that mutant areas involving 1% or more would
probably not escape detection even on an agouti background. The mosaics included in the present
study were recovered during a period of several years by many different observers. Unfortunately, no estimate was made of the proportion of f u r affected, but it is likely from the qualitative
descriptions that this was at least 20%.
The presumed c-locus mosaics were mated to cCh/cch and/or c/c. Wherever feasible, a maximum number of progeny was obtained from such matings in order to derive an estimate of the
percentage of the germline involved in the mosaicism. Stocks were subsequently set up from the
c*/cch (or c*/c) progeny as described elsewhere for whole-body mutants (RUSSELL,RUSSELLand
KELLY1979).
RESULTS
Of 119 presumed c-locus mutants observed in specific-locus experiments with
external radiation, 15 were diagnosed as fractionals on the basis of phenotype
and progeny; and one, which had evidently occurred in the prior generation, on
the basis of progeny alone (a large cluster). These 16 fractionals were distributed
about evenly among controls and irradiated groups (RUSSELL,RUSSELLand
KELLY 1979). In order to arrive at a more accurate measure of relative frequencies, we have summarized numbers of animals observed in externally
irradiated and control groups of all Oak Ridge specific-locus experiments to date,
regardless of whether or not individual ones produced c-locus mutations. Table 1
143
ALBINO-LOCUS REGION O F THE M O U S E
TABLE 1
Distribution of c-locus fractional mutations among externally irradiated and
control groups in Oak Ridge specific-locus experimenis
Sex of
wild-tvpe -parent
__
Male
Female
* Excludes Cr
Wild-type parent irradiated
No.
No.
O~~SDI-~IIZ
c-fractionals
- -
1,718,W
759,750
Parents not irradiated
No.
c-fractionals
No.
offspring
- -
7
1
979,387
165,013
7+
0
(see text) since mutation occurred in previous generation.
shows how the c-locus fractionals are distributed among the major groups. It is
clear that there is no radiation induction of this type of mutation in either sex,
and that the fractionals observed in irradiated groups may thus be assumed to
have a spontaneous origin. A similar conclusion had been derived earlier
(RUSSELL 1964) on the basis of fractionals at all loci observed in a subset of
specific-locus experiments.
Whole-body c-locus mutants were found to be distributed over seven broad
phenotype-viability categories: albino-like, viable (ev)
; intermediate allele,
viable (c?) ;albino-like, subvital (es)
;intermediate allele, subvital (cZs) ;albinolike, lethal (cat)-preimplantation, early postimplantation, or neonatal (RusSELL, RUSSELL and KELLY 1979; RUSSELL and RAYMER 1979). The
c-locus fractionals, on the other hand, were found only in the nonlethal classes
with the following distribution: 10 ev,four czv,and one cas; one was albino-like,
but not tested for homozygous viability and is designated c5.
Over 2000 offspring were classified from the 16 fractionals that had clear germline involvement, ranging from 45 to 402 for individual probands. These progeny
data are presented in Table 2 together with two ratios. The first of these is
designed to provide some indication of whether or not the c* allele is selected
against. Since c* presumably arose from C by mutation, their combined transmission (c* plus C ) should constitute about one-half of the total progeny, unless
there is selection. As shown in Table 2, (C c * ) / ( C cch c2) was indeed
close to 50% for the total set, and is not widely divergent from 50% for any of
the individual fractionals, indicating that there was no major selection against
the new mutations either gametically or in the progeny.
The second ratio is designed to give an indication of the proportion of the gonad
that is made up of mutant tissue. Since, in the absence of selection against c*, the
cchallele is presumably transmitted with 50% frequency from both the mutant
(c*/cch) and nonmutant (C/cch) prtions of the gonad, the proportion of c*-bearing progeny in the total non-ccJ1progeny will give an indication of the makeup
of the gonad. As shown in Table 2, this percentage varied widely for individual
fractionals, ranging from 11.7% to 91.2%, but was reasonably close to 50% for
the overall sample.
+
+ +
144
L. B. RUSSELL
TABLE 2
Progeny of c-locus fractional mutants
Transmissionratios (%)
No. of progeny with
the following allele:
Mutant
ca* 0 18DT
cav 8 24UTh
cav 8 6R75VH
cav 0 17R250M
cav 8 3R250H
cav 0 17FUFo
ca- 8 44PB
C a v a 43UTh
@* 0 28ThP
caw 8 28FrTh,
c*v 8 84CoS
ccv 8 10R30L
czlf 8 58UT
d v 8 7FrS,
cas 0 135G
caw
8 Cr
Totals
C
30
65
128
3
37
37
4
34
33
10
48
38
10
14
10
(316)
501
CGh
C*t
35
9
17
17
31
6
10
17
24
10
27
24
61
11
37
19
86
320
w
14.F
48
38
49
24
66
53
29
75
103
35
62
21
846
c + C*
c + C C h + c'
52.7
56.2
50.2
41.5
53.1
49.0
46.7
46.8
444.8
56.1
49.0
40.0
37.5
45.1
58.0
4Q.3
C*
c + c'
23.1
20.7
11.7
91.2
14.0
21.3
81.0
41.4
23.3
73.0
33.3
61.6
52.4
72.5
65.5
46.7$
45.811
-f c* is used here as a generalized symbol for the mutant allele: CY,
@ or Cas type (see text).
$Calculated as c*/(one-half total progeny), since this mutant is c*/C///C/C (see text), and
the allele that mutated cannot be distinguished from the rest of the genotype.
$ Excludes progeny of Cr.
'11 Average of percentages in this column.
DISCUSSION
Since there was no evidence for the radiation induction of fractional c-locus
mutants, all 16 c-fractionals, including those derived from an irradiated parent,
are considered to be of spontaneous origin. There were only two whole-body
c-locus mutants in the control groups of various experiments (RUSSELL,
RUSSELL
and KELLY1979). In addition, it may be estimated from relative frequencies that
another three or so whole-body mutants in the irradiated groups could have been
of spontaneous origin. Thus, it appears that, at least at the c-locus, the majority
of spontaneous mutations are fractionals.
The mouse embryo does not stem from all of the cleavage products of the
zygote. It is formed from part of the inner cell mass (ICM) of the blastocyst,
which in turn derives from a random assortment of blastomeres that happen to
be on the inside of the morula (HERBERT
and GRAHAM1974). An event, prior to
that stage, that results in two types of blastomeres-mutant and nonmutanttherefore does not automatically produce a mosaic embryo: some embryos will
assort wholly wild type, some mosaic, and some whole-body mutant. The smaller
the number of blastomeres set aside to f o r m the embryo, the relatively larger the
proportion of embryos in the end classes (whole-body mutant or wild type). The
absolute frequency of fractionals observed thus underestimates the frequency of
ALBINO-LOCUS REGION O F THE MOUSE
145
mutational events that result in two types of blastomeres, and the relative frequency of fractionals in the total array of mutants also underestimates this
frequency since some of the whole-body mutants could be derived from such
events.
Taking, however, those fractionals that are observed, one may draw certain
conclusions from their progenies. Unless there is positive selection for mutationbearing cells, the maximum average progeny ratio that can be obtained is 50%
mutant; and this ratio is possible only if embryos were set aside as random assortments of cells from a cell population that, in aggregate, was half mutant (c*/cch)
and half nonmutant ( C / c C h )A. smaller proportion of mutant cells in this overall
blastomere population wmld lead to the production by fractionals of an average
progeny ratio less than 50% mutant (though not exactly corresponding to the
original proportion of mutant cells among those from which the embryos were
assorted, due ta the fact that the end-groups of the binomial, i.e., the whole-body
wild-type and mutant classes, are not being sampled). For example, if the embryo
proper is indeed formed from as few as three ICM cells (MINTZ1970), then the
set from which perceived c-locus mosaics are derived would have to consist either
of one mutant and two wild-type cells o r of two mutant and one wild-type cell.
The former of these groupings would greatly predominate if the probability of a
mutational event was the same at each cleavage division (as suggested for the
case of unstable mutations by SEARLE
[ 19781 ) ,since, under such conditions, the
overall cell population from which the assortment of three was drawn would be
considerably less than 50% mutant. Only if the embryo is set aside from exactly
two cells does the above reasoning not hold. Hotwever, the number is likely to be
greater than three, rather than less (MINTZ1970).
The conclusions concerning the average progeny ratio are independent of the
number of cells involved in the secondary assortment that takes place when the
gonad primordium is being set aside some time after the embryo has started its
growth. That this secondary assortment also involves a small number had already
been concluded from various lines of evidence. [Thus RUSSELL
( 1964) estimated
“around 5”; MINTZ(1968) estimated 2 to 9; and SEARLE’S(1978) data fit well
with these estimates.] The wide spread in individual progeny ratios observed for
c-locus mosaics is in accord with these conclusions.
Since we found the auerage progeny ratio to be close to 50% for the c-locus
fractionals that were observed, it may be concluded that these mice came from
an overall blastomere population that was one-half mutant. (The possible c-fractionals not included in this sample-see MATERIALS AND METHODS-were an
unbiased group, and their inclusion would therefore probably not alter the
result.) A half-mutant blastomere population could result from mutation (C+c* )
in one strand of the gamete DNA, or in a daughter chromosome derived from
pronuclear DNA synthesis of the zygote, or in one blastomere of the two-cell
embryo, prior to replication.
A mutation in one strand of germ-cell DNA would result in a potential mosaic
only if it occurred postmeiotically. The postmeiotic stage in the female is virtually
146
L. B. RUSSELL
nonexistent (since oocyte meiosis is not completed until after sperm entry) ;
whereas, the postmeiotic stage of the male is of several weeks’ duration (spermatid, spermatozoon). This might account for the fact that fractionals were detected
more frequently when the wild-type parent was the male. The occurrence of
spontaneous mutations in one strand of the “already existing” gene was proposed
by MULLER,
CARLSON
and SCHALET
(1961) for Drosophila. If, on the other hand,
the bulk of the mutations that produced the observed fractionals occurred during
pronuclear DNA synthesis, or pre-S in one of the first two blastomeres, one would
have to conclude that these stages (which occupy only a very small fraction of a
generation time) are preferentially susceptible to spontaneous mutations, and
that the male genome is more mutable at these times.
Since mosaics have so far not been induced by irradiation of postmeiotic stages
of the male, it may be suggested that radiation causes too coarse an insult to be
confined to only one strand of the DNA. (However, it should be noted that the
number of progeny observed following postmeiotic irradiation is o n l y a fraction
of the overall historical control population.) Whether mosaics can be induced by
other mutagens in mice is still questionable. Recently, MALASHENKO
(1976) has
reported the presumed induction of three c-locus fractionals by diethylsulfate in
spermatogonia. However, unless the mutagen has a delayed effect, gonia1 treatment is not expected to yield fractionals. Furthermore, results of the genetic tests
of these animals were inconclusive: no new allele was transmitted, and if the
probands mere, in fact, c-locus fractionals, the mutation in all three would have
had to be of the type C + cCh,the rarest of all c locus mutations observed in our
experience [RUSSELL, RUSSELLand KELLY 19791. No attempt was made to
determine whether homozygotes were viable.
LITERATURE CITED
HERBERT,
M. C. and C. F. GRAHAM,1974 Cell determination and biochemical differentiation
of the early mammalian embryo. Cnrr. Top. Develop. Biol. 8 : 151-178.
MALASHENKO,
A. M., 1976 The investigation of the mutagen effect of small doses of diethylsulfate in laboratory mice by the method of specific loci. Genetika 12: 163-165.
MINTZ, B., 1968 Hermaphroditism, sex chromosomal mosaicism and germ cell selection in
allophenic mice. 8th Bien. Symp. Animal Reprod. J. Anim. Sci. 27: 51-60. __ , 1970
Gene expression in allophenic mice. pp. 15-42. In: Control Mechanisms in the Expression of
Cellular Phenotypes. Edited by H. A. PADYKULA,
Academic Press, New York and London.
MULLER,H. J., E. CARLSON
and A. SCHALET,
1961 Mutation by alteration of the already existing
genes. Genetics 46:213-226.
RUSSELL,L. B., 1964 Genetic and functional mosaicism i n the mouse. pp. 153-181. In: The
Role of Chromosomes in Development. Edited by M. LOCKE,Academic Press, New York.
-, 1971 Definition of functional units in a small chromosomal segment 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.
ALBINO-LOCUS REGION O F THE MOUSE
147
RUSSELL,L. B. and G. D. RAYMER,1979 Analysis of the albino-locus region of the mouse. 111.
Time of death of prenatal lethals. Genetics, in press.
RUSSELL,L. B., W. L. RUSSELLand E. M. KELLY, 1979 Analysis of the albino-locus region of
the mouse. I. Origin and viability of whole body and fractional mutants. Genetics 91:
127-139.
SEARLE,A. G., 1978 Evidence from mutable genes concerning the origin of the germ line. In:
Genetic Mosaics and Chimeras in Mammals. Edited by L. B. RUSSELL,Academic Press, New
York and London.
Corresponding editor: D. BENNETT