Zeitschrift fiir Vererbungslehre 92, 165--182 (1961)

M.R.C. Induced Mutagenesis Group Institute of Animal Genetics, University of Edinburgh A CHEMICALLY-INDUCED V A R I E G A T E D - T Y P E P O S I T I O N E F F E C T I N T H E MOUSE By B~vc~ M. CATTA~ACH* With 3 Figures in the Text

(Received May 12, 1961) Introduction I t is now a well-established fact that the effect of a gene m a y be dependent upon its position with respect to neighbouring genes. There are many examples of this position effect (STugTWVA~T 1925) in Drosophila and there is also a similar case in Oenothera (see review by L~.wIs 1950). Most of the position effects are a consequence of rearrangements involving heterochromatic and euehromatic regions of chromosomes and result in a type of mosaicism for the effects of genes lying close to the breakage point. Such position effects have been denoted as variegated--or V-type position effects (LEwis 1950). Until recently examples of this phenomenon have been restricted to Droso. phila, Oenothera, and perhaps also maize (MCOLINTOOK1956), but recently a case of a V-type position effect has been described in the mouse (RussELL and BA~G~A~ 1959). The inheritance of a brown-mottled condition in female mice indicated that such animals were heterozygous for a reciprocal transloeation between the X-chromosome and an autosome (linkage group VIII), the break in the autosome being close to the "brown" locus which carried the wild type allele. A similar case involving another autosome (linkage group I) was also described. I n the present paper evidence for a further example of a V-type position effect in the mouse ~11 be presented. This case resembles those of RVSSELL and BAIqG~A~ (1959) in that a transloeation between the X-chromosome and an autosome is involved, but differs in several features which make it worthy of particular study.

Origin and First Crosses The first mutant animal appeared in a mutation experiment in which the chemical triethylenemelamine (TEM) was employed as the mutagenie agent. The specific loci method, described by RUSSELL (1951, 1952, 1956), and CARTER et al. (1956) was used for detecting mutations. Treated wild type CBA males were crossed with a series of PCT stock females that were homozygous for seven recessive genes [non-agouti (a), brown (b), pink-eye (p), chinchilla (cch), short-ear (se), dilute (d) and piebald or pied (s)], and the F 1 were classified for visible mutations i.e. dominant mutations, or alleles of any of the seven reeessives. The dose of TEM administered was 0.2 mg TEM/kg body weight. The m u t a n t animal, which was a female, appeared in the F x of such a cross. I t was denoted "/lecked" (/d) as its phenotype was that of a white variegated pattern on an otherwise wild type coat eolour. The animal was conceived eight * Medical Research Council Staff.

166

BRve~M. CATTANACg:

days after TEM had been administered to the father, and thus was derived from a spermatozoon that had been mature at the time of treatment. The appearance of the variant animM in an 1~1 generation suggested that either a dominant mutation or a m u t a n t allele of one of the seven reeessives in the PCT stock had been produced. The test-rantings, shown diagrammatically in Fig. 1, were therefore carried ont.

Cross 1. The original/d female was back-crossed to a wild type CBA rome. 37 offspring (22 ~ ~, 15c~c~) were produced in seven litters. All were wild type. The m u t a n t character cannot therefore be inherited as a dominant, but m a y be Mlelic with one of the colour genes such as pied (s). Cross 2. 15 F 2 males were intercrossed with 18 F 2 females, and it was found that the variant character reappeared in crosses of 4 males with 8 females. Four Pll PC~?

_ _

CzT~__I ~ (TREATED)

flecked~.

CBA <~ I

i

F3 PCT & & L FAg. 1.

Diagrammatical

|

I

ftecked ~.~_ |

I

I

|

~

|

I

i'epresentation

J

I

flecked ~_$ L of t h e crosses c a r r i e d i n h e r i t a n c e of ]cl

out

PCT& & |

to

determine

; ?~he m o d e

of

males proved to be completely sterile. The following information was also obtained. Only females exhibit the character, there was no disturbance of the sexratio, and the character was found in combination with all the recessive genes except pink-eye and chinchilla. That is to say, the non-white portions of the coat o f / d animals never showed the pink-eye or chinchilla phenotypes. The results suggest that ]d is sex-limited, but t h a t / d is not allelic with pied or any of the other genes apart from, possibly, pink-eye and chinchilla. The absence of any pceh/d animals can be partly explained by the fact that pink-eye and chinchilla are closely linked [14 crossover units ( G ~ ] s ~ and DICKI~ 1959)], and together produce an almost white phenotype on which the flecked phenotype would not be classifiable. Crossovers between the two genes did, however, occur but in no instance was the variant character seen in combination with either gene. Some connection b e t w e e n / d and the two genes was therefore suspected. Cross 3. 19 F~ males and 8 1~2 females were backcrossed to PCT animals. Of these, two males and four females produced/d daughters. Pink-eye and chinchilla segregated in all rantings, but no crossovers between the two genes was observed among the progeny of those animals which p r o d u c e d / d offspring. The results indicate that both pink.eye and chinchilla are associated with the occurrence of/d, and that a rearrangement involving the pink-eye and chinchilla loci is probably the cause of the lack of crossing over between the two genes. The results further show that males as well as females are capable of transmitting the character. This means that mMes with the equivalent genotype t o / d females do not exhibit the flecked phenotype.

167

A chemicMly-inducedvarieg~teddype position effect in the mouse

Cross 4. ~lecked femMes, obtained in crosses 2 and 3, were backcrossed to PCT males, and the cross was repeated with t h e / d daughters that were produced. As in the previous crosses,/d was found in combination with all the recessive genes except pinE-eye and chinchilla, and no crossing over was observed between these two genes. The results therefore complement the results of the previous crosses. The classification of the progeny of this cross is illustrated in Table 1. Several peculiarities are apparent: Table 1. The cross o/]d #males with PCT ~ m

92 ~

Matings

~

over two generations

Av. Classification of offspring Sexratio littersize o/do ~cch other q. d'd' I ~~ other a~ birth

~

I

~ross 4/d 9 2 • PCT 3~. 9 Next gen./d 29 • P C T ~ .

Tot~l

17 12

48 1 : 1.11 27 1:1 75 I 1:1

g,66

34

20

4al

19

0

27

0

751 0

a) There were twice as m a n y / d as pc +h females. b) There were more pc ch t h a n wild type males. c) There were more pc eh males than pc r females, although the overall sex ratio was normal. d) The litter size was low (el. 6.5 for PCT stock). The following hypothesis was set up to explain the observed results, and evidence consistent with the hypothesis has been obtained. The originM [d female was heterozygous for both pinE-eye and chinchilla and either a reciprocal or nonreciprocal translocation between an autosome (linkage group I) and the Xchromosome such that a piece of autosome, bearing the wild type alleles of pinEeye and chinchilla was transposed to the X. I n the reciprocal form this m a y be shown diagrammatically as x ~ - I pcCh

I +P+ co~~

.

X

Of the two unbalanced

zygotes that are produced when this type of animM is crossed with a normM male, one is inviable

I p~h"

X

deficient for a piece of autosome while

the other, bearing the transposed piece of autosome as a duplication, m a y survive I pcch I/~c ch

I ~-P~ CCh

~ ) .

X'~

Females of this type with two complete autosomes

bearing the recessive genes are also [d. Two types o f / d females are thus produced, the balanced chromosomal type, and the unbalanced chromosomal type with the autosomal duplication. The two equivMent types of male are wild type rather than flecked and a proportion of them are sterile.

Genetieal Evidence for the Flecked Translocation GeneticM evidence for the/lecked translocation was obtained by: A. Crossing flecked females with albino males and test crossing all the types of progeny. B. Crossing/lecked females with pinE-eye males. C. Testing for linkage between the/d-transposition on the X and sex-/inked marker genes.

168

BKUO~M. CATTANACH:

A 1. Flecked/emales • albino males Both the pink-eye and the chinchilla loci have been postulated to be involved in the rearrangement. Proof that this was so for the albino/chinchilla locus was obtained by crossing/d females with albino (ec) males. The albino males employed in this cross were from an inbred stock (JU) maintained by Dr. D. S. F ~ c o ~ E ~ of this department. Table 2. The cross o/ ]d /emales with cc males No. No. No. '.emales litters

Sex-ratio af~ birth

Av. litter size

Classification of offspring

offspring classi-

Type I

/d (c) ~

fled

Type II

Type III

ee~a

/d {ccoh) ~9

Types IV

(c) 9~

29

Types I + II

Types llI

~c~

+43

Type IV

(c) c~

The results of the cross are summarised in Table 2. Two types of ]d daughters were phenotypically distinguishable; one had white markings, while the other had markings of the ccch coat colour. The white flecked type could be distinguished from the N mothers by the fact that the hair in the flecked areas was pure white, while that. of the N mothers was dark at the base as in pe ~h homozygotes. In addition patches of what appeared to be the ceeh heterozygote coat colour could often be seen in the [d mothers. All the recessive type progeny, both f a d e and female, were phenotypically ecch heterozygotes. GENOTYPE MOT )~

PARENTS

GENO T Y P E

PttENOTYPE

fd(flcCh)

+c +c

fd Cc)

+c

pc ~

TYPE x i

+c

Pc~a

~++

TYPE ~

tvcCa"

~

TYPE~

OFFSPRINg +c

f d (cCeh) CC ch

+c

+c--

~

TYPEI V (c)LETHAL

-I-.i-

.tic +c

~ ch

fl ces +c +C

pc c'~ ,

++

pcC--E pc ch OFFSPRING

,

TYPEg fd~pcChc~)

q-c

~ ~

+

TYPE g

+

TYPE$I

Ccch i

TYPE1V (c) LETHAL

I NORHAL

+c +c

DAUGHTERS

++

pcCh

SONS ++

TYPEg

cc i L

+

+c

+c

Rcch

++

TYPE l

FATHER

riCOTHE R

P,4REHTS

CC

SONS

DAUGHTERS +-I-

PHENOTYPE

FATHER.,,,~NORMAL

TYPE~g-

co ch

pcCh

"'7 TYPE~

ccch

+c

Fig. 2. T h e cross of ] l e c k e d f e m a l e s • a l b i n o males. The straight lines represent the a n t o s o m e s o~ linkage group I a n d the w a v y lines the x-chromosomes

Fig. 2 diagrammatically illustrates the hypothesis to explain the observed results and suggests terminology to classify the various types of animals produced. It is to be Presumed that the ]d female parents might be of two kinds as explained above. The two types are therefore shown in the upper and lower parts of the table, and it will be seen that they lead to different expected results.

169

A chemically-induced variegated-type position effect in the mouse

The absence of any albino offspring from this cross indicates that Type IV animals are inviable. This faet, together with the occurrence of two phenotypie classes o f / d females, explains the excess o f / d over recessive type daughters. I t can be seen that a 2 : 1 ratio o f / d to recessive type daughters are expected in litters of T y p e I mothers, but a i : 1 ratio is expected in litters of T y p e I I mothers. I t m a y be presumed that both types o f / d mother were represented in the present cross. This would account for the number o f / d daughters being in excess of the recessive type though not being twice as frequent. I n Fig. 2 both Type I and Type I I males have been shown as wild type for there is no reason why, if one is wild type i.e. Type I, the other should not also be so. The expected ratio of 2 : 1 wild type to recessive type males is then in contrast with the observed numbers. This could be explained b y reduced viability of one or both of the wild type classes, but it would not explain the excess of cc~h males over cc sh females, for these should occur in equal numbers. The g 2 test of equality is 4.9 which has a probability of between 2 % and 5%. The excess of cech males was, however, not observed in later crosses and was probably due to chance despite its formal significance. The results of this cross are compatible with the hypothesis but do not completely discriminate between the two types of /d female parents. Tests on the progeny of this cross were therefore carried out. A 2. Test crosses o / t h e progeny o / / d (pc oh) ]emales

Only progeny of females which produced a~ least one ]d (c) daughter were selected for test because such females are presumed to be of Type I and should produce offspring representing all of the different genotypes postulated. a ) ]d (c) ]emales. Eleven/d (c) females were crossed with pc eh (PCT) males and four with ce (JU) males. The results arc recorded in Table 3. Table 3. The cross o/ Type I / d (c) #males with pc ch males, and the cross o] Type I / d (pCoh) :emales with outbred cc males Classification of offspring

Phenotype of sire

No. a n d t y p e of 9 9

pcCh (PCT) 12/d (c) ce (JU) 4/d (e)

g z

Sexratio a t birth

5"6 Z

Type I

Type II

Type III

Type IV

99

99

??

99

18

0

34 1:1.03 4,59 123 17(1)'20 14 1:0.79 5.0 39 14 Total 16/d (c) 48 1:0. 4.71 I 162 51 (1)* 14/d (pech) cc (BC) 41 1:094 5,12] 187 29 135 * One exceptional female was wild type in phenotype.

9 27

33

?

[ 0

Type I Type Type and III IV III

45

22

0

9

7

?

54 I 29 55 135

? 0

The following points about the results of this cross are important : A good 2 : 1 ratio of ]d to recessive type females was obtained, and again phenotypieally distinguishable ]d females were produced. There was an excess of wild type over recessive type males, and this m a y be attributable to the fact that in this cross all the mothers were of Type I. Some of the wild type tomes were noticeably

170

B~vc~ M. CAT~x~AC~:

small in size and they frequently did not survive to weaning. This suggests t h a t one of the two theoretical classes of wild type males is relatively inviable. No pc ch or cc, but only ccch recessive t y p e offspring were produced when pC ch males were employed. This indicates t h a t none of t h e / d (c) mothers were hcterozygous for pink-eye or chinchilla. The litter size was low and this provides supporting evidence for the occurrence of inviable classes. All these points are in accord with the hypothesis t h a t t h e / d (c) mothers are of Type I. Table 3 also shows the results of the equivalent cross in the following generation. Type I / d (pc oh) females were crossed with cc males from an outbred stock (BC), maintained b y the author, in .order to t r y and increase the fertility of the flecked stock. The data were in close agreement with those o f the previous generation, but although the litter size was somewhat larger, the proportion of wild t y p e males decreased slightly. One exceptional female was produced in this t y p e of cross and this will be discussed later. b) fd (ccoh) /emales. The ]d (ccoh) females were crossed with either pc eh or ce males. I t was found more convenient to use cc males, for some /d(cChc ca) were produced when pc ch males were employed, and these were difficult to classify on certain backgrounds. Table 4 records the results of this t y p e of mating. The important points in these results a r e : / d and recessive type females appeared in equal numbers, and Table 4. The cross o/Type ! I ]d (ccch) ]emales with either pceh or cc males No, females litters

23

at birth

5411:0.76

size

5.81

offspring classified

TypeII~

274[74

T y p e I I I 9 9 I TypeIIc73 T y p e I I I c ~

85

23

92

two phenotypically distinguishable classes of b o t h / d females and recessive type males and females were obtained b y segregation of the albino and chinchilla alleles in the heterozygous mothers. There was an extreme shortage of wild t y p e males when classification was done at three weeks of age. The examination of litters at an earlier age showed t h a t these males were present in higher numbers at birth, but they did not grow as fast as their sibs and m a n y died before they reached weaning age. The litter size was larger t h a n t h a t of the ]d (c) females, indicating t h a t no lethal class was produced. These points are in accord with' the hypothesis t h a t t h e / d (ccoh) females are of Type I I and arc heterozygous for the albino and chinchilla alleles carried on the two complete autosomes. I t is apparent t h a t the Type I I males, which are the only t y p e of wild t y p e male produced b y Type I I females (see Fig. 2), are a relatively inviable class, and this explains the shortage of wild type males from the expected numbers in the previous crosses. An important point about all the results presented so far is that not one crossover between either the pink-eye or the chinchilla locus and the break point has yet been detected. e) Recessive type ]emales and males. 15 ceeh females and 21 cecJ* males were crossed with ~cch or cc animals. The results are presented in Table 5. At least

171

A chemically-induced variegated-type position effec~ in the mouse

20 offspring were scored f r o m each cross, a n d all were of t h e two recessive t y p e s e x p e c t e d if t h e p a r e n t s possessed t h e n o r m a l c h r o m o s o m e c o m p l e m e n t a n d were h e t e r o z y g o u s for albino a n d Table 5. The cross o / T y p e I I I ccch females and males chinchilla. These animals therewith either pc eh or cc animals fore represent TypeIII of Fig. 2. No. Av.size litter Sex-ratio d ) W i l d t y p e mares. T h e Sex tested No. tested offspring at birth classified wild t y p e sons of b o t h T y p e I a n d T y p e II females were female . . 15 223 7,2 1:1.01 tested. Fig. 3 diagrammaticmale . . 21 323 7.3 1 : 1.26 ally i l l u s t r a t e s t h e e x p e c t e d results of t h e crosses of t h e t w o t y p e s of wild t y p e males w i t h cc females. I t c a n be seen t h a t l i t t e r s of T y p e I males can be d i s t i n g u i s h e d f r o m t h o s e of T y p e I I males b y t h e f a c t t h a t T y p e I males p r o d u c e one i n v i a b l e class of son. The effect GENOTYPE

P/RENTS

+c +c

MOTHER

PHENOTYPE

NORMAL,

cc

GENOTYPE FATHER ++ TYPE I "")'

~ §C

OFFSPRING + c

+c

4--I-

TYPEI

TypE ~

+C

f d (C)

fz(~

" C- "+

.,,.2

§C

MOTHER NORMAL,

cc

+c

fdCcc)

+c

DAUGHTERS -+c- z

§ OFFSPRING pcCh ,

_

§

_

+.

~

f-/ THER ++

pc dz

+c

TYPE IV (c)LETH,4L,

. ~ rvze ~

+C

+c D'dRENTE

+

SONS

D`4UGHTERS ++

PHENOTYPE

~

TYPE [E

+

~

TYPEI~1

cc

SONS

~ - ~ TYPE H

§

+§

cc ch

. . . . . _ -

TyPE~ , fd(~c~)

~ +C

r m z~

Fig. 3. The cross of flecked-bearing wild t:cpe males • albino females. The straight lines represent the autosomes and the wavy lines the sex-chromosomes

can be seen in t h r e e w a y s : T y p e I males p r o d u c e o n l y one p h e n o t y p i c class of m a l e offspring, t h e sex-ratio is a b n o r m a l , a n d t h e l i t t e r size is reduced. T h e results of t h e crosses are p r e s e n t e d in T a b l e 6 : t h e y are in a g r e e m e n t w~th t h e h y p o t h e s i s set o u t in Fig. 3. T y p e I males could be d i s t i n g u i s h e d from Table 6. The cross o / T y p e I and Type I I males with cc #males r

Genotype of males ~

Type I

Type II

[

No

I l

1~o ~ off r: ~p mgi

79 I 3

"

t

~

t

I 4.8

: . offspring e

I 357

r

.

] 214 I

Classification of offspring . . ~ $s 3~ Others

4

I 137

I

1

I l~ 0

Type II males by the classification of their litters for litter size and sex-ratio, and also by the fact that they produced only one phenotypic class of son. Type I daughters of Type I males could also be distinguished by breeding tests. Since t h e t w o t y p e s of wild t y p e m a l e could be d i s t i n g u i s h e d b y genetical tests, i t was possible to show t h a t T y p e I I females p r o d u c e o n l y T y p e I I sons,

172

B~vcE M. CATTA~Ae~:

while Type I females m a y produce both types of wild type sons. D a t a to illustrate this fact is presented in Table 7. A point of note is t h a t although the wild type sons of Type I females were taken at random for testing, Type I I males were only one third as frequent as Type I males. Also, the Type I females produced a number of completely sterile sons. This class of male possibly belongs to Type I and represents a more extreme case of reduced fertility, for no sterile sons were produced b y Type I I females. I n support of this conclusion is the fact that m a n y Type I males become sterile after producing only one or two Table 7. The classification of the wild type sons litters. I t would thus appear t h a t o/Type I and Type l I #males the fertility of this class of male is No. ] Classification of sons very variable, and there is a tendency T y p e of Wild type Mothers sons t e s t e d Sterile for complete sterility to occur. The results in Table 7 therefore indicate t h a t only about 25% of II 22 2 0 Type I I males are viable. This is in agreement with the estimate of viability based on the ratio of wild type to recessive type males in the litters from which the test animals were selected. I t is also in agreement with the estimate of viability of Type I I males in litters of Type I I females (see Table 4). I n order to illustrate the small size of the Type I I males at three weeks of age, the individuals of some litters of both Type I and Type I I females were weighed. The average weights of the different types of offspring are shown in Table 8. I t can be seen t h a t the average weight of Type I I sons of Type I I mothers was Table 8. The average weights o/ el#print o/ crosses o/ Type I and Type I I #males with cc (BC) males Type II ~

Type I 9

T y p e of Mother

T y p e I I I ~ ~;

W i l d T y p e c73

Type III I

No.

Av. wt. gins

No.

av. W t . grns

No.

Av. Wt. ffms

43

9.8

No.

Av. W t . gms

No.

5.3

42

I Av. Wt. [ gms

J

I III

[ 26 -

-

8.3 -

-

84

7.9

9.7

appreciably lower than t h a t of their sibs. I t is apparent, however, t h a t although small Type I I males were produced by Type I mothers, the proportion that survived to three weeks of age was not large enough to lower appreciably the mean weight of the compound wild type class. There was an overlap of the weights of the "small" and " n o r m a l " wild type males, and the two could only be distinguished within litters. With only nine exceptions all the female progeny of both types of males were ]d and all the sons were recessive types. The close linkage of the/d-transposition with the X-chromosome is good evidence for the theory t h a t the X is involved in the transloeation. The occurrence of the nine exceptional animals will be discussed later. B. Flecked/emales • pin]c-eye males The pin]c-eye as well as the chinchilla locus has been postulated to be involved in the rearrangement. This is indicated in /d (pc c1~) females where the flecked

A chemicMly-inducedvariegated-type position effect in the mouse

173

markings are the white colour of the pc ~a compound homozygote. The absence of crossing over between the two loci i n / d animals provides additional evidence. FinM proof that the pin/c-eye locus is involved in the translocation was obtained by the cross o f / d females with pp males. The pp mMes employed were from the I~F stock maintained by Mrs. CLAYTO~ of this department. The stock had the pin/c-eye gene on an otherwise wild type CBA background. T h e / d females used were obtained from the early crosses o f / d females with pc ch males (see Table 1), and Table 9. The cross o/#males /lec/ced /or could have been either of Type I or Type II. Table 9 presents the results of crossing both pin~c-eye and chinchilla with pp males s i x / d females with pp males. A new type ~ 1 Classification of o~fspring of /d femMe was produced for the flecked / 1 1 1 ~eema,les / ana_/or I l ~ ? ~ t + <~<~ I ~P ~' ~' markings were of the pin/c-eye coat colour. All the recessive animals were of the pin/ceye phcnotype. The pin/c-eye locus must therefore be affected by the rearrangement. An interesting feature about t h e / d (p) animals was that on the average the flee]ced markings were smaller than those of t h e / d (e) o r / d (ceoh) animals.

1;

I

1,0 I ]7

C. Tests/or linkage between the/d-transposition and sex-linked genes The cross of/d-bearing males with cc females has provided evidence that the /d-transposition is attached to the X-chromosome. I t was considered that tests for the linkage of ]d with sex-linked marker genes would provide conclusive proof that the X-chromosome is involved in the translocation, and would locMise the/d-transposition on the X-chromosome. The two sex-linked genes Tabby, (Ta) and Bent-tail (Bn), were selected; these are separated by a distance of 11 crossover units (FALCONEtr 1954). The Table 10 The lin/cage o//d and Ta Non-crossovers Phenotype

Crossovers No.

/d~?

138

Ta/-/- ; recessive

147

~ype ~

61

Ta; recessive type ~c~

140

Phenoty-pe

ma!+; ld ~ ?

No.

Crossing over %

1.43 ?

recessive type o~

Ta 3 3

4.69

recessive type ~c~

3.45

results of the hnkage tests o f / d and Ta are shown in Table 10. The method employed was to cross/d females with Ta;cc or Ta;ce ch males to obtain Ta/-t-; /d daughters. These were then crossed with cc males and the progeny were scored for crossovers. I n practice it was found that only three of the four classes of offspring could be classified, for the Ta/-t- phenotype could not always be distinguished on the albino background. In addition some difficulty was encountered in classifying/d females with extreme flecked markings for the Ta/-t- phenotype. This meant that only the males could be accurately scored for crossovers. The

174

BRve~M. CAT~AXXC~:

results show t h a t /d and Ta are closely linked, the crossover frequency being approximately 4 %. D a t a for the linkage of ]d and Bn have not yet been obtained, but a few preliminary crosses o f / d females with Bn males were made to see if a n y hemizygous Bn females could be produced. This would indicate t h a t the translocation was reciprocal in nature, and t h a t Type I I animals were deficient for the piece of the X-chromosome bearing the Bn locus. 570 such hemizygous Bn females were produced but unfortunately it was not possible in this cross to distinguish phenotypically those females which possessed the /d-transposition. I t is unlikely, however, t h a t the B~ locus is involved in the translocation.

Cytological Evidence for the Transloeation Genetical evidence for the presence of an X-autosome translocation in the

flecked stock has been obtained b y the progeny tests on/d-bearing males, and b y the linkage of /d with Ta, as explained in the previous sections. Cytological confirmation of the translocation was therefore sought as follows. Males bearing the/d-transposition were tested b y the method described b y SLIZY~XSKI(AvEg~AcH and SLIZY~SKI 1956; FALCOnEr, SLIZY~SXI and A v ~ A C ~ 1942). The examination of meiotic I metaphase plates of both Type I and Type I I males did not reveal a n y evidence for the presence of a translocation. No ring-of-four structures were seen and 20 bivalents were regularly counted. The analysis of pachytene chromosomes b y Dr. SnIzY~sxI has, however, provided cytological evidence for the a t t a c h m e n t of a piece of autosome %0 the end of the X distal to t h a t which associates with the Y. Both genetical and cytological evidence thus indicate t h a t a piece of autosome has been transposed to the X. Sterile wild type males produced b y Type I females were also examined cytologically. Spermatozoa were produced, but the sperm heads showed gross structural, abnormalities. This was probably the cause of the observed sterility.

Intererosses of ld Females and/d-Bearing Males An a t t e m p t was made to obtain females t h a t were homozygous for the X bearing /d-transposition. Type I I females were therefore crossed with Type I and Type II males. Fifty-six offspring were obtained from the intercross of Type II animals. Of these 25 were/d females, 8 were wild type males, and 23 were recessive type males. All appeared perfectly normal. 22 of the/d females were crossed with cc males, and all p r o d u c e d / d and recessive type daughters and wild type and recessive type sons. The appearance of the recessive types indicates t h a t the /d mothers possessed a normal X-chromosome, and therefore were not homozygous for the transposition. The results suggest t h a t females, homozygous for the transposition and with two normal autosomcs, are inviable. Evidence in support of this conclnsion is the low average litter size of the intercross mating (3.3), and the excess of males, in spite of the fact t h a t the wild type males are of Type I I and thus only 25 % viable.

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The cross of Type I I females with Type I males has to date only yielded one litter as a consequence of sterility problems with the Type I males. The mother of this litter was genotypically/d (cc) and the father was heterozygous for pinkeye and chinchilla. The litter contained one exceptional female that was phenotypically wild type, one/d (c) female, one/d (ccoh) female, and two wild type males. I t is conceivable that the exceptional female is homozygous for the transposition, and this may also apply to the /d (c) female for it showed only slight flecked markings. Both females have been crossed with cc males. The exceptional female produced 15 ]d daughters and 15 wild type sons ; t h e / d (c) female produced 3/d daughters and 2 wild type sons. All the daughters were flecked for the albino coat colour so it is likely that both females were of Type I. The absence of any recessive type progeny provides supporting evidence that the two females are homozygous for the transposition. I t may therefore be that only Type I females homozygous for the transposition are viable, while the Type I I homozygotes are inviable 1

Exceptional Animals The cross of /d-bearing males with cc females normally produces only ]d daughters and recessive type sons. Nine exceptional animals appeared in crosses of this type. Six of the exceptional animals were recessive type females and two were phenotypically /d males. The occurrence of these classes of exceptional animals has already been reported (CATTa~AC~ 1960). I t WaS shown that the recessive types were matroclinous X 0 females for they did not possess their fathers' /d-bearing X-chromosomes, and the males were X 2 Y in chromosomal constitution having inherited the /d-bearing X- and Y-chromosomes from their fathers. The occurrence of t h e / d males has been attributed to non-disjunction of the X- and Y-chromosomes in the meiotic divisions of their/d-bearing fathers. This explanation could also account for the appearance of the recessive type females ; alternatively the loss of the Y- or/d-bearing X-chromosomes could have occurred shortly after fertilisation (see I~VSS~LL and SAYLO~S 1960). The ninth exceptional animal produced by a /d-bearing male was a phenotypically wild type female. Its phenotype could be explained in two ways: I t could possess one autosome bearing the wild type alleles of pink-eye and chinchilla as a result of a crossover between the/d-transposition and the complete autosomc, i.e. I +~ +~k I+~ c

I pc0h

. Alternatively, this exceptional animal could be a patro-

clinous XO female, for t~*:SS~LL and ]~A~GI~A~a(1960) have shown that the variegation caused by one of their position effects is not expressed in XO females. The present exceptional animal m a y therefore have arisen as a consequence of nondisjunction in the gametogenesis of the mother, or shortly after fertilisation, or it could have occurred as a result of the mother herself being an XO female that Several wild type females have now been produced in the cross of Type II females with Type I males and are in the process of being tested geneticMly. The results to date indicate that they are homozygous for the/d-transposition while their ]d sibs all appear to be heterozygous for the transposition. It can therefore be concluded that the /d phenotype is not expressed in females in which both X-chromosomes carry the transposition. Z. Vererb.-Lehre, Bd. 92 12

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Bgvc~M. CA~A~AC~:

h a d arisen s p o n t a n e o u s l y . Chromosome counts of t h e m o t h e r h a v e been carried out, using t h e m e t h o d of F~c~I~WlM~ (1960) for corneal e p i t h e l i a l tissue, a n d 40 c h r o m o s o m e s were r e g u l a r l y counted. The m o t h e r was n o t therefore a n X O female. Genetical tests on t h e e x c e p t i o n a l female are now in progress, a n d one l i t t e r has been p r o d u c e d so far in a cross w i t h a Ta; cc male. The l i t t e r consisted of t h r e e females of which two were Ta/+;/d a n d one was p h e n o t y p i e M l y Ta/O; cc 1. I t is therefore m o s t l i k e l y t h a t t h e e x c e p t i o n a l a n i m a l r e p r e s e n t s a f u r t h e r e x a m p l e of p r i m a r y n o n - d i s j u n c t i o n a n d is a p a t r o c l i n o u s X O female. One o t h e r e x c e p t i o n a l female a p p e a r e d in t h e cross of a T y p e I ]d(c) female w i t h a pc ch male. This female also was p h e n o t y p i c a l l y wild t y p e . I t s occurrence could a g a i n be e x p l a i n e d either b y a crossover b e t w e e n / d - t r a n s p o s i t i o n a n d t h e c o m p l e t e a u t o s o m e , or b y p r i m a r y n o n d i s j u n c t i o n , this t i m e i n v o l v i n g t h e loss of t h e p a t e r n a l sex-chromosome. B r e e d i n g t e s t s y i e l d e d no useful i n f o r m a t i o n for o n l y one l i t t e r was p r o d u c e d a n d this was e a t e n a t birth. H o w e v e r , m i t o t i c c h r o m o s o m e counts on coded slides i n d i c a t e d t h a t t h e female possessed o n l y 39 chromosomes. T h e m o s t l i k e l y h y p o t h e s i s for t h e occurrence of t h e e x c e p t i o n a l a n i m a l is, therefore, t h a t i t was a m a t r o c l i n o u s X O female. I t w o u l d seem therefore t h a t t h e flecked p o s i t i o n effect is analogous t o t h a t d e s c r i b e d b y RUSSELL a n d BA~G~IAM (1960) in so far as v a r i e g a t i o n is n o t e x p r e s s e d in X O females. A n a t t e m p t was m a d e to d e m o n s t r a t e t h a t X O females w i t h t h e / d - t r a n s p o s i t i o n are wild t y p e b y m a k i n g a cross t h a t should p r o d u c e this t y p e of female. A T y p e I m a l e was crossed w i t h Ta/O;cc females. To d a t e o n l y one l i t t e r of t w o females has been p r o d u c e d . One female was wild t y p e in p h e n o t y p e a n d m a y be a n X O female w i t h t h e f a t h e r ' s X - c h r o m o s o m e b e a r i n g t h e / d - t r a n s p o s i t i o n 2; t h e o t h e r female was of a n u n e x p e c t e d p h e n o t y p e for i t was Ta/O;cc r a t h e r t h a n / d or wild t y p e . The o n l y e x p l a n a t i o n to a c c o u n t for t h e a p p e a r a n c e of this a n i m a l is t h a t i t m u s t be a m a t r o c l i n o u s X O female, caused b y ~ f u r t h e r incidence of n o n - d i s j u n c t i o n . B o t h females are being f u r t h e r i n v e s t i g a t e d .

Discussion E v i d e n c e is p r e s e n t e d to show t h a t a mosaic coat colour in t h e mouse, d e n o t e d flecked (/d), r e p r e s e n t s a n e x a m p l e of t h e p h e n o m e n o n of v a r i e g a t e d - t y p e posit i o n effect. T h e flecked p h e n o t y p e is caused b y a t r a n s l o c a t i o n b e t w e e n a n a u t o s o m e (linkage g r o u p I) a n d t h e X - c h r o m o s o m e . I n this r e s p e c t /lecked is a n a l o g o u s w i t h t h e t w o p o s i t i o n effects described b y Russ~LI~ a n d BA~GItA~ (1959, 1960), a n d t h e t h r e e cases i n d i c a t e t h a t t h e X - c h r o m o s o m e is p a r t i c u l a r l y effective in p r o d u c i n g t h i s t y p e of p h e n o m e n o n . 1 Mitotic chromosome counts have now been carried out on the Ta/O; ca daughter of the presumed patroclinous XO female and 39 chromosomes were regularly counted on coded slides. I t seems safe to conclude that it possessed an XO chromosome constitution and this, together with the genetical evidence, indicates that the mother is a patroclinous XO female which owes her origin to primary non-disjunction either in the gametogenesis of the mother or in the fertilized egg. 2 The wild type female produced in the cross of a Ta/O;cc female with a Type I male has now been shown to have an XO chromosome constitution and to carry the /d-transposition on its single X-chromosome. This provides suppor$ing evidence for the hypothesis tha$ a normal as well as a/d-bearlng X-chromosome are required for the expression of the flecked phenotype.

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I n the present case the rearrangement involves the transposition of a piece of autosome0 bearing the wild type alleles of the linked genes ~pink-eye (19) and chinchilla (ceh), to the X-chromosome. Females possessing the transloeation and which are heterozygous for either, or both, of the reeessive genes exhibit the variegated phenotype. Males t h a t possess the same chromosomal constitution are wild type. This type of animal is referred to as Type I. When Type I animals are crossed with normal animals four types of zygote are produced, and two of these have unbalanced chromosomal constitutions. One of the ehromosomally unbalanced types is deficient for a piece of autosome and is inviable. The other possesses two complete autosomes and carries the piece of autosome transposed to the X as a duplication. Females of this class are viable, and when both their autosomes bear the reeessive genes they are flecked in pheno type. Males of this class are only partially viable and are wild type. Both genetical and cytological evidence in favour of these conclusions have been obtained. The complete inviability of one of the zygotic types is indicated b y the absence of one phenotypic class of offspring in litters of Type I animals and the associated low litter siae. The viability of Type I I females, and hence the occurrence of two genotypic cIasses o f / d daughters in t/~ters of Type t females, is indicated by the observation t h a t two phenotypic classes are distinguishable in suitable crosses, and by the fact twice as m a n y / d females as recessive females are produced. Proof that two types of ]d females occur was obtained by breeding tests. Litters of Type I I females differ from those of Type I by the production of [3 and recessive type daughters in equal numbers, an extreme shortage of wild type sons all of which can be shown to be of Type II, and by larger litter sizes. Evidence for two types of wild type males in litters of Type I mothers is more difficult to obtain for the two classes cannot be accurately distinguished phenotypically, and the ratio of wild type to recessive type males is usually short of a 2 : 1 ratio. The occurrence of two types of wild type males is, however, indicated phenotypieally by the size difference within this class in litters of Type I females, but proof t h a t two types of males occur was obtained by breeding tests. Type I males possess the complete translocation and eonseqnently half their sons o b t a i n the deficient autosome and are inviable. Litters of Type I males therefore differ from those of Type I I b y the absenee of one phenotypic class of son, an abnormal sex-ratio, and b y low li~ter sizes. The deviation from a 2 : 1 ratio of wild type to recessive males is a consequence of the reduced viability of T y p e I I tomes. Evidence for the poor viability of Type I I males is dearly demonstrated in litters of Type I I females, since these produce only one class of wild type son, i. e. Type II. These should be produced in equal numbers with the recessive type males, but they are markedly deficient at three weeks of age. Small size and a tendency to die at an early age are characteristic features of Type [ I males for only 25--30% survive go three weeks of age. Compagible with this observation is the fact t h a t the average litter size of Type I I females is lower than that of their normal sibs (el. Tables 4 and 5), and this m a y indicate that some Type I I males die prenatally. Breeding tests on the progeny of Type I females also indicate the reduced viability of Type I I males. Type I females should produce Type I and Type I I 1"9,*

]78

BRUCE M. CATTANACH:

sons in equal numbers, but genetical tests on all the wild type sons produced by this class of female is a given period proved t h a t most are of Type I. A proportion of these sons were sterile, and as no sterile sons of Type I I females have yet been found, it was concluded t h a t they represented a more extreme example of the reduced fertility of Type I males. If the sterile males are included as being of Type I, then four times as m a n y Type I as Type I I males are represented at maturity. This again indicates t h a t only about 25 % of Type I I males are viable. There is one discrepancy concerning the viability of Type I I males. The observed ratio of wild type to recessive type males in the total litters of all Type I females is closer to a 2 : 1 ratio than would be expected if only 25 % of the Type I I males survive. However, a greater proportion of Type I I males m a y survive with supplementary feeding, and there is some evidence t h a t fewer survive in larger litters (cf. generations in Table 3). If this is so, then it would be expected t h a t a greater proportion would survive in litters of Type I females which produce somewhat smaller litters than Type I I females (cf. Tables 3 and 4). The reduced viability of Type I I males m a y mean t h a t the original translocation was of a reciprocal type. Proof t h a t a piece of autosome was transposed to the X-chromosome is provided b y the transmission of the flecked character from father to daughter, and b y the linkage o f / d with the sex-linked gene, Ta. There is no such direct evidence t h a t a piece of X was reciprocally exchanged in the transloeation and attached to the autosome, but the difference in viability between Type I and Type I I males m a y indicate t h a t this was the case. If Type I males possess both reciprocally rearranged chromosomes and thus possess the full chromosome complement, then they would be expected to be fully viable, which they are. Type I I males, on the other hand, would then be deficient for a piece of the X-chromosome and their loss of viability would be expected, since it is known t h a t the 0 u chromosome constitution is inviable (W~LsHo~s and RvSSnLL 1959). I t is most likely therefore t h a t a piece of the X-chromosome was rearranged when the original translocation was induced and was probably reciprocally exchanged with the ]d-transposition. Several observations indicate t h a t the rearrangement ihvolves only small pieces of the chromosomes. The viability of one of the two unbalanced chromosome types indicates t h a t the autosomal duplication does not produce noticeable deleterious effects and, as has already been discussed, a deficiency for a piece of X could not be very large or it would cause lethality in the male. In addition no sign of an association of four could be seen in the cytological preparations. This suggests t h a t the transposition is too small to create pairing difficulties between the rearranged chromosomes and their homologues. The present example of the V-type position effect closely resembles those of ReSSEr~T. and BA~CC~HA~r(1959, 1960) but it differs in several features. They are similar in that an X-autosome translocation is the cause of each of them, but a most important feature which distinguishes the inheritance of ]lecked is the survival of one of the urrbalanced chromosome types. Another distinguishing factor of/lecIced is the fertility of most of the males that are heterozygous for the transloeation. The sterility that occurs in some is not a consequence of a brake-

down of meiosis at pachytene as occurs in I~USSELL and BA~GHAM'S mice but is due to abnormal sperm production. I f the meiotic breakdown in I~USSELL and

A chemically-induced variegated-type position effect in the mouse

179

BA_~OI~AM'Smice is caused by pairing difficulties, then the fertility of/d-bearing males m a y again indicate the small size of t h e / d transposition. A further factor which distinguishes the flecked position effect is that crossing over does not appear to occur between the marker genes and the break point on the transposition, and the complete autosome. This is especially interesting in view of the fact t h a t the position effect concerns two genes, pink-eye and chinchilla. The loci of these genes are 14 crossover units apart (GI~wE~ and DIc~:IW 1960), but no crossing over was observed to occur between either gene and the break point. The absence of crossing over in/d-bearing mice could be explained in two ways. I t is possible that the break is close to one of the genes and the rearrangement suppresses crossing over between the two genes. A more likely alternative is t h a t crossing over is prevented by an absence of pairing between the normal antosome and the transposition as the cytological data suggests. In either case it would seem thet the crossover distance of 14 units represents only a small length of actual chromosome. I t has been observed t h a t ]d (pcoh) animals occasionally show two variegated coat colours for, in addition to the white markings of the combined pc ch genes, markings resembling the colour of the cech heterozygotes have been found. This occurrence could be explained in two ways. If the pink-eye allele is transferred to the complete autosome by somatic crossing over, variegation for only the chinchilla gene could take place. I t is unlikely t h a t this hypothesis is correct, however, since meiotic crossing over does not appear to occur. An alternative hypothesis is t h a t a spreading effect is operative. The phenomenon of the spreading effect has frequently been observed in Drosophila where it has been foumd that the effect exerted by heteroehromatin on genes on a newly adjoining euchremarie region of chromosome spreads along the chromosome from the break point always affecting first the gene closest to the break point. Thus variegation for the gene nearest to the break point m a y often be seen independently of t h a t of the neighbouring genes. I n / d (pcch) animals, the colour of the additional variegated markings was very similar to the coat eolour of ccch heterozygotes. This suggests t h a t the cCh/O is phenotypically like ccch and t h a t the albino locus is nearer to the break point than the pink-eye locus. The observation that on the average the variegated markings of ]d (p) animals are smaller than those o f / d (c) animals is in agreement with this conclusion. The break point and the pink-eye locus thus seem to lie on opposite sides of the albino locus. I t m a y be added t h a t the same conclusion would be reached on the hypothesis that somatic crossing over is the cause of the cc~h variegation in ]d (pc oh) females. While the spreading effect indicates an analogy between the V-type position effects in Drosophila and the mouse, there is at least one difference. I n Drosophila, these position effects are expressed in both sexes, although the Y-chromosome tends to suppress variegation (see review b y LEwis 1950). I n the mouse, however, variegation is normally only expressed in the female. This could be attributed to a suppression of variegation by the Y-chromosome, or to the presence of only one X in the male. The data presented here, together with those of R~rSS~LL and BA~GHA_~I(1960) and CATTA~CH (1960), indicate t h a t the number

180

BRYCE M. CATTA~ACH:

of X-chromosomes is the decisive factor. Thus XO females and X Y males possessing the transposition are wild type, and X X females and X X Y males with one X bearing the transposition are variegated. This difference between Drosophila and the mouse m a y be correlated with the difference in the sex-determining systems in the two animals. A further difference between the position effects in the mouse and Drosophila will emerge if the wild type female or the slightly flecked female produced b y the cross of a Type I I female with a Type I male are proved to be homozygous for the/d-transposition 1. I n Drosophila, homozygotes for rearrangements t h a t cause V-type position effects tend to show more variegation. I t has been suggested t h a t this m a y possibly be due to the closer association of heterochromatin with the affected pair of alleles in the homozygotes since pairing of the homologous segments of the rearranged chromosomes m a y be easier (LEwis 1950). If female mice homozygous for the /d-transposition should be non-variegated or show reduced variegation, this could be explained on the hypothesis t h a t it is unlikely t h a t both rearranged genes in the homozygote will be affected in any one cell. On the other hand, it is perhaps of some significance t h a t m a n y of the sex-linked genes in the mouse produce mottled phenotypes, and t h a t the mottling is only produced when the genes are in the heterozygous condition. I n this respect the V-type position effects in the mouse closely resemble the sex-linked genes. The mottled phenotypes caused by V-type position effects and sex-linked genes in the mouse possibly indicate t h a t the function of genes associated with the X-chromosome are modified by the proximity of the heterochromatin of the X. An alternative explanation for the tendency of sex-linked genes in the mouse to give mottled phenotypes has recently been put forward by LYON (1960). She postulates t h a t it is the usual and normal thing in the somatic cells of the female mouse for one X-chromosome to be inactivated. I n a female heterozygous for a m u t a n t gene some cells would have the m u t a n t gene active and others the normal, and this would result in a mottled phenotype. On this hypothesis cells in which the/d-bearing X is active will give rise to the wild type coat colour, and those in which the normal X is active, the recessive type coat colour. In this manner LYoN's hypothesis explains why X X females and X X u males with only one X-chromosome bearing the/d-transposition are/d, while XO and X X females and X Y males whose X's bear the transposition are wild type. With this explanation, the variegated phenotypes caused by X-autosome translocations in mice are not analogous to the V-Type position effects of Drosophila. I-Iowever, if the operation of a spreading effect correctly explains the occurrence of the two variegated colours i n / d (pceh) animals, then LYon's hypothesis does not hold and the two phenomena are probably analogous. The cytological evidence has indicated t h a t the piece of autosome transposed to the X is attached to the end of the X opposite to that which associates with the Y. Linkage tests are now being carried out w i t h / d and sex-linked marker genes. Once /d has been localised with respect to these genes, it should then be possible to localise on the linkage m a p the end of the X t h a t associates with the Y. I See Footnote 1, p. 175.

A chemicMly-induced variegated-type position effect in the mouse

]81

The finkage of the /d-transposition with the X-chromosome has allowed the classification of non-disjunction products among the offspring of /d-bearing males, and nine exeeptionM animals have been produced. The occurrence of phenotypieally flecked males and recessive type females has already been reported, and has been attributed to non-disjunction of the X- and Y-chromosomes in the fathers, at least in the eases of the males (CArrTANACI~1960). The single wild type female which appeared in the same type of cross could not have arisen by non-disjunction in the father for it possessed its father's/d-bearing X-chromosome. Although mitotic chromosome counts have not yet been carried out, alI the genetical evidence indicates t h a t it represents an example of primary nondisjunction and is a patroelinous XO female 1. This type of X 0 female has been induced b y irradiation shortly after fertilisation ( t ~ u s s ~ L and SAYzog 1960), and the occurrence of three exceptional females has been explained on the hypothesis t h a t they possess only the paternM X-chromosome ( M c L A ~ 1960, CATTA~ACI~ 1960). The present example, however, probably represents the first substantiated example of a spontaneously occurring patroclinous XO female.

Summary Evidence is presented to show that a chemically induced variegated coat colour mutation in the mouse, denoted flecked (/d), is caused by a transloeation between an autosome and an X-chromosome and represents an example of a V-type position cheer. Two autosomal genes, pink-eye and chinchilla, are affected b y the rearrangement. The expression of/d is normally limited to females, but studies of the character in NO females and X X Y males have shown t h a t the determining factor is the number of X-chromosomes. Two types o f / d females occur: one has the complete transloeation, and the other has an unbalanced chromosome complement possessing the transposed piece of autosome as a duplication. Both appear to be fully viable. Two equivalent types of ]d-bearing males also occur: the balanced type is fully viable but tends to be sterile, the unbalanced type is fully fertile but only about 25 % are viable. The loci of the two affected genes are 14 crossover units apart but crossingover between t h e m is suppressed. Cytological studies on/d-bearing males have indicated t h a t this m a y be due to a lack of pairing between the transposition and the complete autosome. Observations are described suggesting the operation of a spreading effect, which would indicate t h a t the chinchilla locus is nearer to the break p o i n t t h a n the pink-eye locus. Several exceptional animals showing unexpected phenotypes appeared in t h e / d stock. Most of these have been shown to be aneuploid for the sex-chromosomes and to have arisen by non-disjunction. The position effect has been compared with those of I~VSS~LL and BANG~A~ (1959, 1960) in the mouse, and also with those described in Drosophila (L~wls 1950). Acknowledgements. My grateful thanks to Dr. C. AVE~AeR, 1%1%. S. for her most helpful advice and encouragement throughout the course of the work, and to Dr. ]3.1Yr. SLIZY~S~:Ifor his help with the cytological aspects of the problem. I would Mso like to thank Dr. D. S. ]?~eONE~ for reading the manuscript and Dr. N. S. F]~C~HEI~ for demonstrating his technique for counting chromosomes. 1 See Footnote 1, p. 176.

182

B~cc~ M. C4TTA~AC~: A chemically-induced variegated-type position effect

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