PSEUDOALLELISM AT THE NOTCH LOCUS IN DROSOPHILA

W. J. WELSHONS AND E. S. \'ON HALLE

Biology Division, Oak Ridge National Laborator) ,I OnA Ridge, Tennessee

Received February 6, 1962

O T H sex-linked dominant and recessive visible mutaiits have been Lwnd at Bthe Notch locus in Drosophila. The Notch ( N ) ini:tant5 itre lethal in 1ieiniLv- gotes and homozygous females (WELSHONS 1958b), and don Jnant in heteroq gotes displaying notched wings, thickened wing veins and n iricir bristle abnor- malities. Since many Notch mutations are associated with a cytologically de- tectable deficiency for at least salivary band 3C7 (MOHR 1919, 1923; DEMEREC 1939; SLIZYNSKA 1938), the correspondence between the loss of band 3C7 and the production of a N phenotype has led to the conclusion that the ~ J C U S is con- tained within this single band (DEMEREC 1939).

In addition to the dominant mutants, there exists at the samr: I ytological loca- tion a series of recessive visibles called facet ( f a ) , split ( ( d ) , facet-notchoid (fa""), notchoid (nd) and facet-notch (fa") (GLASS 1933, not used in these studies). The two mutants fa and spZ have distinguishably different rough eye phenotypes (GLASS 1933; BAUER 1943), while the recessive visibles fa"" (BAI -ER

1943) and nd (obtained from M. J. FAHMY) have notched wing phenotypes asso- ciated with thickened wing veins. Consequently, the phenotypes of nd/nd, fano/ fano, and N / + flies are superficially similar (all wing mutants) but differ markedly from the phenotypes of fa/fa and spZ/spZ (eye mutants) animals.

If a heterozygote is made of a dominant N that is known to be deficient for the single salivary band 3C7, D f ( l ) N , and a recessive visible, fa for example, the Df ( I ) N / f a heterozygote has the expected notched wing phenotype because of the expression of the dominant N mutant; in addition, the rough eye phenotype of the recessive fa mutant, is observed (MOHR 1923). This pseudodominant ex- pression of the recessive mutant, present as a single dose in the heterozygote, is to be expected if the deficient N chromosome lacks the normal allele of fa. Similarly, one observes the pseudodominant expression of the recessive eye mu- tant spl in Df(l)N/spZ heterozygotes. The expression of the mutants facet- notchoid (fa"") and notchoid ( n d ) when heterozygous with a N is more difficult to assess since the phenotypes of all three are, to begin with, somewhat similar. Heterozygotes of N and nd have extremely notched strap-like wings, and their viability and fertility is greatly reduced. The combination N and fa"" is almost completely lethal, the survivors having a phenotype resembling the Notch- notchoid heterozygotes. The increased severity of the abnormal wing phenotype is due, presumably, to the pseudodominant expression of the recessive visible superimposed upon the expected expression of the dominant N .

loperated by Union Carbide Corporation for the U. S. Atomic Energy Commission.

Genetics 47 : 743-759 June 1960

744 W. J . W E L S H O N S A N D E. S. VON H A L L E

In the above examples, deficiency N’s were intentionally chosen for demon- stration; however, many dominant Notch mutants are now available which are indistinguishable from point mutations by cytological and/or genetic evidence, although, by phenotypic criteria, they are inseparable from deficiency-Notches. Thus, in heterozygotes of “nondeficiency” Notches with the recessive visible mutants, one again notes the pseudodominant expression of the recessive mutant even though it can be shown that its wild allele is carried on the Notch-bearing chromosome. This is a pertinent observation when one later tries to interpret the nature of dominant nondeficiency Notch mutants.

The pseudoallelism of fa, fano, and spl has already been reported WELSHO ON^ 1958a,b). These three visibles were shown to be pseudoallelic to certain non- deficiency dominant N’s and therefore pseudoallelic to each other, although trans- heterozygotes of the recessive visibles failed to show a clear-cut mutant pheno- type. It was also reported that certainly two, and possibly three, of the dominant N’s were pseudoallelic since they could be separated by crossing-over, and as- signed a linear order relative to each other and relative to the visibles fa, fano, and spl. Seeing that dominant N’s may be alternatively referred to as recessive lethals, we critically demonstrated their pseudoallelism ( WELSHONS 1958b). In this case, the trans-heterozygotes were noncomplementary (lethal).

For the presentation of additional information, it is convenient to look upon the Notch locus as a complex composed of two pseudoallelic systems, a recessive visible series superimposed upon another of recessive lethals (dominant N mutants). The separation of the two is, of course, artificial, but it is a great aid in the approach to an interpretation and analysis of the complexity of the locus.

When comparing pseudoallelic loci in Drosophila and microorganisms, one often finds such dissimilarities that it sometimes seems necessary to conclude that the organization of genetic material is not the same. One of the most obvious differences encountered is that within a complex locus in Drosophila there seem to be few mutable sites, each with many alleles, while in microorganisms there appear to be many more mutable sites, most having relatively fewer allelic mutations. However, regardless of the limited data presently available at the Notch locus, there is the suggestion that pseudoallelic recessive lethals map as do the mutants of microorganisms. Furthermore, a consideration of the two pseudoallelic arrays at this locus, first independently and then in combination, leads one to a logical explanation for the lack of correspondence encountered when making comparisons with various other organisms.

MATERIALS

Pseudoallelism of three recessive visibles used in these experiments has already been described. The fourth is discussed here.

The dominant Notch mutants require some additional discussion. The mutants NZ64-40 and NZ64-103 , of X-ray origin, were obtained by DEMEREC (see BRIDGES and BREHME 1944) and subsequently shown upon cytological examination to be nondeficient and free of aberrations. The mutants NCO (WELSHONS 1956) and

NOTCH LOCUS 745

Nl*4 are of spontaneous origin, the latter mutant having been detected as a spon- taneous mutation to N on a chromosome which already carried the fa pseudoallele (N3*4 was called N** in an earlier publication, WELSHONS 1958b). N"'", of X-ray origin, was obtained in this laboratory by B. NICOLETTI. The remaining mutants, NfiohZ1, N60114, NEOflO, and N"Ql1 are part of a group of mutants produced in low- dose gamma irradiation experiments by P. T. IVES and sent to the authors for investigation. The N s of IVES and the one of NICOLETTI were not examined cytologically. It was tacitly assumed that the N chromosomes were nondeficient and free of aberrations when the wa marker, 1.5 units to the left, was crossed on to the chromosomes without obvious difficulty. This recombinational criterion, which has been used by other investigators to detect, genetically, the presence of chromosome aberrations (GREEN and GREEN 1956; GREEN 1959a; CARLSON 1958), has been reliable when applied to the Notch locus. Of the five N s screened in this way, all have subsequently recombined with at least one pseudoallele of Notch, and three of them (NNlc , N60f10, N 6 O g l 1 ) have recombined with two differ- ent pseudoalleles.

Three of the N ' s are atypical in some respects. The mutant NCO is more prone to have thickened wing veins than notched wings (WELSHONS 1956), and, in addition, the mutant phenotype is enhanced by the presence of a duplication for the locus Dp(l ; l )Co, while the phenotype of all other N s tested by us is sup- pressed by the duplication. N*64-10S7 with a mild mutant expression, mottles for split when heterozygous with this recessive visible pseudoallele (see BRIDGES and BREHME 19M), and NE0Q1' is associated with a dominant temperature-sensitive rough eye abnormality.

METHODS A N D RESULTS

With the exception of a single cross, all flies were counted in a Drosophila counter (KEIGHLEY and LEWIS 1959). This machine was constructed at Oak Ridge from blueprints kindly sent to us by E. B. LEWIS.

The term Notch will generally refer to those pseudoalleles which can be characterized as dominant visibles and/or recessive lethals. The N symbol has been shortened for practical purposes, i.e., Ne64-4O will be symbolized as N-4". When there is little chance of misinterpretation, the term Notch will also be usacl to refer to the whole complex of visibles and lethals.

It was shown previously that the order and recombination frequency of three of the recessive visibles frcm left to right on the X chromosome was as follows: fa to fano, 0.05 percent; fano to spl, 0.03 percent (WELSHONS 1958 a,b). To this series it is possible to add the mutant nd. The position and recombination fre- quency were obtained from the cross of & spl/nd rbQ Q by U@ fanospl rb8 8 (Table 1). In a total of 38,900 tested chromosomes, six waspl nd rb, and five + + 4- + recombinant chromosomes were recovered, so that it is possible to place the locus of nd 0 028 map units to I he right of spl (Figure 1 ) .

As a prelude to finding a more precise genetic location of a given recessive lethal (a dominant Notch mutant), N/spZ females, heterozygous for closely linked peripheral markers, were used in an attempt to recover recombinants

746 W. J. W E L S H O N S A N D E. S. VON H A L L E

TABLE 1

Recombinants obtained between the pseudoalleles N and spl, N and fa, and spl and nd. Parental males were fa fano in the N/fa cross. In all other cases, parental males were wa fa11o spl rb

Constitution of parental females

Recombinant Number Percent chroniosonies tested recombination

W" + Nflo +

+ + s p l + rb

6-uQ spl nd rb

5-+ + + + 3-+ + + +

12-Y + + rb

4-y w a + + rb*

10-wa + + rb

2-& + + rb

7-y W" + + rb

3-y w a + + rb

38,900

42,482

41,041

35,523

13,612-t

38,575

48,445

46,226

0.028

0.007

0.029

0.01 1

0.073

0.005

0.014

0.007

* Inrludes two questionable recombinant females, which died and could not be progeny tested + Only male progeny were used.

0.05 fano 0.03 SPl 0.028 nd I f 4 I I

0.023 0.007 I 0.003 0.016 0.029 I

N-109

0.011

FIGURE 1 .-Map of the Notch locus. Numerals above the line indicate map distance between recessive visibles. Numerals below the line indicate recombination frequencies obtained between a N and spl . NQll not separated from nd.

between the two mutants, and at the same time, to localize the lethal either to the right or left of spl. In these preliminary experiments, autosomal inversions were not used to increase the frequency of recombination, because it is possible by proper manipulation of the cross to obtain sufficient resolution for these initial tests; furthermore, when the recombination frequency is once obtained in this manner, it has considerable prognostic value in future crosses.

NOTCH LOCUS 747

Two examples will suffice to clarify the origin of the data presented in Table 1. From females of the genotype w" N N t c +/+ spl rb mated to wa fa"" spl rb males, three recombinant chromosomes, of the type + + + +, were recovered out of a total of 42,482 tested. In view of the close linkage of the markers apricot (w") and ruby ( r b ) (wa-spl = 1.5 percent; spl-rb = 4.5 percent), their origin is best explained as the result of a single crossover event between NN%c and spl, the mutant N N t c occupying a position to the left of spl. Since the mutant is lethal in the hemizygous males while the heterozygous combination NNic/fano is lethal in females, only half the progeny are expected to survive. The reciprocal recombi- nant, Wa N N i c spl rb, is expected to be lethal in males, and one would expect it to be lethal in females heterozygous for fano. In any event, the reciprocal ex- change has never been detected and since only one of the two products of a cross- over can be recovered, and only half of the tested chromosomes survive, the recombination frequency is equal to 0.007 percent (3/42,482).

When y Ng" +/+ spl rb females were mated to Wa fano spl rb males, 12 recom- binant chromosomes of the type y + + rb were recovered (y-spl = three percent recombination) out of a total of 41,041 tested. In this case, it appears that Ng" lies 0.029 units ( 12/41,041) to the right of spl.

A consideration of the results obtained in the above two crosses already suggests that NN" and NgZ1 are separable mutaut sites since one is localized to the left of spl while the other lies to the right. Furlhermore, since it has already been shown that N-4O lies approximately 0.02 units to the left of spl (WELSHONS 1958b), while the above cross indicates that NxIc lies only 0.007 units to the left, one is able to estimate the size of an experiment designed to separate the two N mutants in later crosses.

It is always dangerous to draw conclusions concerning the localization and separability of mutant sites on the basis of their comparative crossover frequen- cies, and the situation is even less satisfactory when these frequencies are based on the recovery of only one of the two possible recombinants. One might argue that in the two cases just described, the double mutant recombinant type could be recovered in female progeny if the parental females were mated to males that did not carry the fano mutant; i.e., wa NN%c + +/+ + spl rb females by wa 4- spl rb males should produce viable w" NN%c spl rb / Wa + spl rb recombinants, and in this way both products of a single exchange between N and spl could be recovered. However, owing to the pseudodominance of spl heterozygous with a N , it is highly likely that wa N spl rb/& 4- spl rb would be so similar in pheno- type to the relatively frequent recombinant resulting from a single exchange between spl and rb, the fl N + rb/& + spl rb female, that the chromosome with the coupled pseudoalleles would not be detected. In other words, neglecting the outside markers for the sake of simplicity, N spl/+ spl probably cannot be differentiated from N +/+ spl, a conclusion which is strengthened by the obser- vation that fa Nla4/fa + females are very similar to f NfIo/fa f or any other heterozygote of N and fa tested to date.

The data of Table 1 indicate that the N's are separable from the recessive visible spl, but the separability of one N from another is indicated only by their

748 W. J. WELSHONS A N D E. S. VON HALLE

gross localization to the right or left of spl, and when two N's lie both on one side of spl, their existence at different mutant sites is indicated only by their comparative recombination frequencies. It is therefore necessary to provide additional evidence that the recessive lethals occupy separable mutant sites.

Obviously, one might demonstrate separable sites by obtaining females of the genetic constitution a N" +/+ NU b (a, 6, x, and y are used as a general notation) looking for recombinants between the two N loci. However, this is not so easily done because the method of synthesis would require mating a female heterozy- gous for one N to a male hemizygous for the other, and N is lethal in males; furthermore, females hetero-ygous for two different N ' s might fail to survive. Fortunately, there exists in Drosophila a duplication for the Notch locus which has been removed from its normal position in the X chromosome and inserted into the second autosome (LEFEVRE 1952). The duplication, symbolized as Dp(l;2R)w""', carries a t least the w+ and N+ alleles. It is possible to obtain viable males of the comtitution a N"+; Dp(l;2R)w5lb7/ + which, when mated to females heterozygous for NU b, produce females a N" + / + NU b; Dp(1;Z) w51h'/ +. If heterozygotes of the two A7's do not appear unless the duplication is also present, W/NY is a lethal combination. Since the Dp (Dp = Dp(1;2R)w5'") can be expected to segregate independently of the X, one can look for recombi- nants between the N s among the nonduplication-bearing progeny.

In this way it was shown that W - 4 n and N C O occupied different mutant sites (WELSHONS 1958b,c). The existence of a third mutant site, NIP), a slight distance to the right of spl and between N-Ln and NCO was suggested on the basis of recom- bination frequencies obtained from NI2& / spl heterozygotes. Its separability from N-4" is demonstrated by the following cross: Females of the constitution y w" fa + NI"+ / + w" + N-4" + rb; Dp/+ were mated to w" fawo spl rb males (the N's are shown linearly arranged in the order predicted from previous crosses), In this case the X's are homozygous for w", in order to determine the presence or absence of Dp, which, in turn, carries U>+. Only half of the progeny are expected to carry the duplication and survive, the remaining half will be lethal with the exception of the wild-type recombinants between the N s , and in this case. the peripheral markers y and rb are used to determine the linear order of N-Ln and NJ24. If W4" is localized to the left of N@4, one should expect to recover among the non-Dp progeny the recombinant chromosomes y w" fa + + rb. Eight such chromosomes plus one nonrecombinant exception were recovered (Table 2) , yielding a recombination frequency of 0.022 percent. One would not expect to recover the double mutant since it is unlikely that it could survive in male or female.

Since NI24 and Nro both appear to be to the right of spl and might be closely linked, one or more autosomal inversions were introduced in an attempt to in- crease the recombination frequency in the X chromosomes. Hence a cross was made of y w" fa NIz4 + + / + wa + + NCO rb; Cy/Dp; / + females by wu fano spl rb males, where the mutant Cy (Curly) is carried on the inverted second autosome (SMI ,Cy) (LEWIS and MISLOVE 1953), and the mutant Ubx19" is carried on the inverted third autosome LEWIS 1952). At the same time, another

NOTCH LOCUS 749

TABLE 2

Recombinants and exceptionals obtained from Nx/NY and N"o/nd heterozygotes. In the cross Nco/N-103, parental males were fa fano sn; Cy/Pm. In the remaining cases parental males

were as follows: I n crosses autosomally Dp/+ and Cy/Dp, males were wa fano spl rb. When the autosome was Cy,Dp/+, males were wa fano sp l rb; Cy/+.

W h e n the autosome was Cy,bwv,Dp/+, males were wa fano spl rb; Cy/Pm

Constitution of ( roc5 parental females

~-

1 y Wa fa + Ni24 + Dp 8-Y .- + wa + N40 + r b ' + I-+

2 y Wa fa NjS4 + + C y Ubx 4-+

+ & + + N C O rb ' D p ' + 2-+

3 y wa fa Ni24 + + cy 7-+ 1-Y

4 y wa + Nhrac + Cy,Dp 17-y

. -. -

.- + ~a + + N C O rb ' Dp 2-+

.- + wa N-40 + rb ' + 5 y Wa + N g l 1 + CY& 7-Y .- + Wa N C O + rb ' + 1-+

y wa N-'OJ+ + ? + 1-Y

f @ + N - I O S + rb 3 + 1-+

6 + Wa + N C O rb Cy,Dp,bwv 7-+ 1-+

7 y W O fa + Ni24 + Cy,Dp,bwv 11-y

-

8 + @ + nd rb Cy,Dp 7-+ .- y wa N C O + + ' +

Recombinants and exceptinnals

No. tested and percent

recombination

wa fa + + rb

wa + + + rb*

w " + + + +

w " + + + r b

w a + + + + Wa f a + + +* wa + + + rb

d + + r b

2><8/71,656

0.022

2~ 4/78,5 74

0.010

2~7 /108 ,902 0.010

17/55,722

0.031

7/116,683

0.006

7/21,230+

0.033

Could come about by a double exchange within the locus , Only male progeny were used.

cross of the same nature was used, but in this case only the SM1,Cy inversion was present. If NCO occupies a site to the right of NJ24, one would expect to recover + wn + + + + recombinant chromosomes. Four such recombinants (plus two nonrecombinant exceptions) were recovered when both inversions were used (recombination frequency = 0.01 0 percent), and seven (plus three nonrecombi- nant exceptions) were recovered when only the SM1,Cy inversion was used (recombination frequency = 0.013 percent).

In the last-mentioned crosses, it was observed that the D p could be inserted into the SM1,Cy chromosome by a double exchange, and it was possible to isolate an SMl,Cy,Dp chromosome. While the frequency with which the duplication was inserted into the SM1,Cy was not accurately measured, it was obvious that

750 W. J. WELSHONS A N D E. S. VON H A L L E

when an additional inversion was present the event occurred much more fre- quently than in its absence; hence, one would expect that once the duplication was inserted into SMl,Cy, it would generally remain there provided additional chromosome inversions were not introduced. The usefulness of this chromosome can be illustrated as follows: In crosses of the type a N" + / 4- NU b; Dp/ + females by & fano spl rb males, only that half of the progeny which possess the duplication can survive with the exception of the wild-type recombinants between the N's, and even these cannot be detected if the duplication is present. As the mutant sites get closer together, autosomal inversions are introduced to increase crossing-over, but in this study, it was undesirable to use more than one inversion because of adverse effects on viability and fertility. The recovery of the SMZ,Cy, D p ch-omosome allows one to make a fortunate compromise coupled with a gain in genetic resolution. One can take advantage of the stimulus to crossing-over afforded by a single autosomal inversion, and, when the mutant Cy, lethal in homozygous condition. is introduced into the parental males, it is now possible to eliminate three quarters of the uninteresting (nonrecombinant) or useless ( duplication-bearing) progeny.

The inverted chromosome with the insertional duplication was used in two crosses, one of which is represented as y @ + NITTc +/ + wa N-4O + rb; SMZ, Cy , D p / + females by wa fan0 spl rb; SMl,Cy/ + males. From this cross 17 y wa + + rb recombinants were recovered, yielding a recombination frequency of 0.031 percent. Similarly, the presumed linear order of NCO and Ng" was verified; the recombination frequency was 0 006 percent (Table 2).

Once it was realized that the SM1 ,Cy,Dp could be used to kill off three quarters of the progeny, the next logical step was to create a cross in which the only sur- vivors would he those which carried the recombinant. As will be shown presently, the SMZ ,Cy,Dp chromosome could be used in this way if it carried an additional mutant which. like Cy, was lethal in the homozygous condition. In a discussion of the topic with E. H GRELL, it was discovered that he had a chromosome which might suffice; it was an altered SM1,Cy on which a brown-variegated (bw") mutation had been induced following X-irradiation. If the Dp could be inserted into this altered SMl-Cy, and if bwv was lethal when heterozygous with Plum ( P m ) , the chromosome could be used €or our purpose. Subsequently, it was found that the D p could be inserted into the altered SMl,Cy, and other tests indicated that while some heterozygotes of bwvaie and Pm would live, the majority died. Recently, it has been found by HOCHMAN (1961) that 60-80 percent of the heterozygotes are lethal.

The altered chromosome, S M I , C ~ , ~ W ' ~ ~ " , Dp, was used to separate N mutants that promised to be very close together. In the cross of +@ + N C O rb /y wp N-lo7 + +; S M _ I , C ~ , ~ W " ~ ' " , Dp/ + females by fa fano sn; SMl ,Cy/Pm males, seven 4- W" + + t- recombinants and two nonrecombinant exceptions were obtained. No attempt was made to measure the frequency of recombination. For our pur- poses, it was enough to verify the separability and linear order of N-'"' relative to NCO.

In the final cross, performed like the one above, the linear order of NI2& and

NOTCH IOCUS 75 1

N-Io3 was found to be the reverse of that predicted from the data in Table 1. Eleven y fa + 7- rb recombinants and one nonrecombinant exception were recovered, indicating a linear order from left to right of spl, N-’OS, ” $ 4 . Since the predicted order obtained from heterozygotes of the N ’ s and spl was based upon only two recombinants in the case of NJ24 (WELSHOKS 1958b), and only two definite recombinants plus two more probable ones in the case of N-lo3 (Table 1) the reversal of order is not too surprising.

In the pseudoallelic crosses listed in Table 1, no nonrecombinant exceptions were detected, whereas many appeared among the progeny of Nx/NU hetero- zygotes. In part, at least, this difference could be a reflection of the greater num- ber of chromosomes tested in the latter experiments; furthermore, nonrecombi- nant exceptions have been previously detected in crosses like those of Table 1 (WELSHONS 1958a,b) so that it is not impossible for them to occur in Nx/spl crosses. The exceptions can be explained either as revertants or as products of multiple exchanges-but since at least one autosomal inversion was utilized as a stimulus to crossing-over in every case except one, an explanation for the origin of at least some exceptions based on multiple exchanges finds some support in this data. Most of the exceptions can be explained by a minimum of two crossovers, one which occurred within the locus and one outside the locus. However, in crosses 1,3, and 7 of Table 2, two exchanges within the locus and within approxi- mately 0.083 to 0.096 map units would be required.

Since nonrecombinant exceptions have always occurred less frequently than recombinants, we do not feel that their occurrence jeopardizes conclusions per- taining to the linear positions of the various pseudoalleles, especially since there is a high correlation between the data of Table 1 and Table 2. From the data presented here and elsewhere (WELSHONS 1958a,b), it is possible to map the locus (Figure 1). With the exception of the recessive lethal Nq”, only those mutant sites which have been demonstrated to be separable from the next adjacent mutant sites have been placed on the map. Thus, there are six recessive lethal and four recessive visible loci-a total of nine distinct loci since Ng” has not been demonstrated to be separable from nd. The recessive lethals Nf’O, Nhel, and NI’&, which appear to be between spl and NCO, are not shown on the map because they could be positioned at this time only by their recombination frequency with the recessive visible spl (Table 1 ) .

DISCUSSION

Pseudoallelic recessive lethals at the Notch locus: The data of Table 1 and Table 2 indicate that some recessive lethal N mutants apparently tend to cluster in a regioa starting a short distance to the right of spl and extending on to NCO. In addition to the mutants of Table 1, there are at least several others which appear to be in this locality but have not been reported here because of insufficient data. Many of the mutants in this cluster might prove to be inseparable and on the basis of negative evidence classified as truly allelic. However, owing to the elaboration of genetic tools such as the SMl,Cy, D p and the altered SMZ,Cy, bwr57e , Dp chromosomes, and because of a limitation imposed by the number of

752 W. J. WELSHONS A N D E. S. VON HALLE

N mutants available, attempts were made to separate some of these, and in every case our attempts were successful (N-lOl, N’244, and NCO, Table 2). Consequently, it is not unreasonable to assume that there are additional separable sites in this region. Furthermore, in the last series of N’s tested against spl, a new position was found to the left of spl (N”lc) , and another a relatively great distance to the right of NCO (Ng”) , indicating the possibility that other recessive lethals will eventually be found in places conspicuously bare at the moment.

The assumption will be made that many more sites within this complex locus are capable of mutating to a recessive lethal condition, and that eventually it will be shown that the array of recessive lethals will not differ materially from an array of mutants at any one of a number of complex loci in microorganisms. The chief merit of the assumption which rests upon the separability of only six N’s is that it will lead to the development of a logical explanation for the apparent discordances frequently noted when comparing pseudoallelic loci of Drosophila and microorganisms.

All the trans-heterozygotes utilized in the separa tion of the N’s (Table 2) were lethal, hence the N combinations of Table 2 are noncomplementary. In addition. all possible combinations of N-4O, NJZ4, N-loS, and NCO have been tested and found to be lethal in trans-heterozygotes. One might then question the cis condition. Since the trans-heterozygotes are lethal, our interpretation of these loci as pseudo- allelic would be jeopardized only in the event that the cis-heterozygotes, N x N i / f +, were also lethal. A lethal cis condition is unlikely since it would mean that coupling two N mutants on one chromosome had created a dominant lethal, while deficiencies for the entire complex at the Notch locus plus neighboring loci as well are not lethal when heterozygous. In a sense, deficiency N heterozygotes serve as an approximation to a viable cis condition which can then be compared to the lethal trans-heterozygotes.

What is the basis for the dominant phenotype seen in both deficiency and non- deficiency N s ? Apparently, for the nondeficiency mutants, the genetic material contained in at least the segment measured from fa to nd is present but inacti- vated, whereas the same genetic material is completely missing in deficiency mutants; hence, both classes of mutants are presumably deficient in enzyme activity and therefore have the same phenotype. Consequently, the trans-hetero- zygotes are lethal because they resemble homozygous deficiencies, whereas the pseudodominant expression of a recessive visible heterozygous with a N is due to the inactivation of the wild-type allele of the recessive visible carried on the N chromosome. This interpretation of the recessive lethal N’s does not differ from that given to the rII mutants of phage (BENZER 1957) and, for example, the pan-2 mutants of Neurospora (CASE and GILES 1960). The Notch locus differs from the loci of Neurospora in one respect-no complementary recessive lethals have yet been detected.

Pseudocrllelic recessive visible loci at the Notch locus: It is instructive to con- sider the recessive visible pseudoalleles without recourse to the information con- tributed by the N’s. Suppose that the recessive lethals were not associated with the obvious and clear-cut. notched-wing phenotype. If this were so “Notches”

NOTCH M C U S 753

would have been recognizable only as recessive lethals, and it is extremely doubt- ful that they would have been singled out for intensive investigation. The clue to pseudoallelism between the rare point-mutation lethals and visibles (i.e., the pseudodominance observed in heterozygotes), if detected, would have been interpreted as the expected consequence of combining a recessive visible with a deficiency for the visible locus. This situation would have encouraged an investi- gator to consider only the recessive visible mutants, and initially, fano and nd, both wing mutants, would have appeared to be allelic on the basis of their heterozygotic noncomplementarity. Subsequently, it would have been possible to demonstrate their pseudoallelic nature. As the result of a careful examination one might have considered the possibility that fa, primarily an eye mutant, was, nevertheless, a pseudoallelic member because heterozygotes of fa with fano and nd occasionally have slight nicks in their wings (as do fa homozygotes). The complementarity of spl and fa, both eye mutants, would have discouraged a pseudoallelic interpretation for these two. There is nothing about spl or hetero- zygotes of spl with fano or nd that would have suggested pseudoallelism; yet spl, so different from fa"" and nd, is localized midway between the two. Since some complementary pseudoalleles (like spl) need not resemble other pseudoallelic members (like fano and n d ) , the discovery of their true relationship would be dependent almost entirely upon fortuitous circumstances, and consequently, the number of detectable mutant sites at a complex locus consisting of only recessive visibles would tend to a minimum.

The complementarity seen, for example, in the heterozygotes of spl with fano and nd is probably due in part to the fact that the phenotypes of the individual mutants are so dissimilar, and it is perhaps not too surprising to observe what is essentially a normal phenotype when an eye mutant is combined heterozy- gously with a wing mutant. Similar situations exist at the d p locus, as noted by MULLER (see CARLSON 1958), and at the y locus ( SANDLER, HART and NICOLETTI 1960). It is doubtful, however, that even in these cases the phenotype is, in a strict sense, normal. In a study of all the mutants at the Notch locus which were avail- able at the time, and before it was known that the mutants were pseudoallelic in the modern sense, BAUER (1943) noted that while heterozygotes of various of the recessive mutants were superficially normal, one could still discern, with a good deal of labor, some subtle differences.

Comparison of Notch wi th Ubx and dp: The Notch locus is similar to the Ultra- bithorax (Ubx) pseudoalleles (LEWIS 1951) in a number of ways. At both com- plex loci, pseudoallelism has been demonstrated between dominant mutants, alternatively classified as recessive lethals, ( N compared to Ubx) and a series of recessive visibles (fa, fano, spl , and nd compared to bx, bxd, and p b x ) . At the time of the last publication on the Notch work, before the mutant nd which is noncomplementary with fano was available, all recessive visibles at the Notch locus were at least superficially complementary; a similar condition exists at the Ultrabithorax locus. Cytological deficiencies are associated with the recessive lethal N condition. This is also the case with U b x but both N and U b x can exist in

754 W. J. WELSHONS AND E. S. VON HALLE

a form which is indistinguishable from a point mutation, and when heterozygous with recessive visibles, pseudodominance is observed.

Notch differs from Ultrabithorax in that six recessive lethal N loci are known whereas only one recessive lethal site has been demonstrated at Ubx. Perhaps the Ubs locus has, in fact, only one or very few recessive lethal sites. Perhaps the discovery of only one site is a reflection of the difficulty of recognizing Ubx reces- sive lethal mutations on the basis of their dominant phenotype. Certainly the dominant N mutants are more striking phenotypically than the dominant Ubs.

The two complex loci differ in another respect. At Ubx a dominant, homozy- gous viable mutant, Contrabithorax ( C b x ) , is known; no counterpart for this mutant has been found at the Notch locus, although it is possible that an Abruptex (Ax)-like mutant which has arisen in the course of experiments might be related to the Notch locus like Cbx is related to Ultrabithorax.

It is interesting to compare the dumpy locus with Notch. At dumpy ( CARLSON 1958) there exists a series of recombinationally separable recessive visible loci, oblique ( ' o ) , vortex ( ' U ) and oblique-vortex ('ou) (see CARLSON 1961, and CARLSON and SOUTHIN 1962). The heterozygotes 'o/'u are wild type, while 'o/'ou is oblique and 'u/ 'ou is vortex. At the same locus there exists a series of recessive lethals, symbolized as '2, '02, 'Zu, ' o h , all of which are noncomplementary (lethal) when combined heterozygously. The symbolism used by CARLSON (1958) allows one to derive the phenotype of any given heterozygote of one of the lethals with one of the visibles, or, for that matter, with a combination of heterozygous lethals. Thus, '2/'2u or any combination possessing two '2's is a lethal; ' 0101 is oblique in phenotype; 'ou/'oZu is phenotypically oblique-vortex, etc. The recessive lethals 2u and 02 can be localized between the same two adjacent recessive visible mutants (CARLSON 1961) but it is not possible to attempt a direct separation of the two lethals since the noncomplementarity of the 2u/ol heterozygotes precludes the experiment, and there is no duplication available that can be utilized to cover the lethal effects of heterozygotes while attempting to recover recombinants between the lethal sites.

The dumpy locus is therefore another example of a complex locus composed of recessive visibles and recessive lethals, and in this case the recessive lethals would not ordinarily be classified as dominant mutants. It should be noted that since heterozygotes of 'olu with the recessive visibles '0, 'ou, and ' U , are phenotypically oblique, oblique-vortex, and vortex, respectively, one can look upon these various phenotypes as the result of a pseudodominant expression of a recessive visible when heterozygous with a pseudoallelic recessive lethal, and in this way contrast the pseudodominance at dumpy with that at Notch. From this point of view, the ' o b recessive lethals resemble the N mcltants since they show pseudodominance for all the recessive visibles, and one might expect by analogy with N that these mutants will eventually be shown to exist at various mutant sites.

The ' 2 , '02, and ' lu mutants cannot be compared to any recessive lethals now existing at the Notch locus, although it should be noted that MULLER and ALTEN- BURG (1921 ) once reported a mutant iacet-lethal ( f a z ) which was not pheno- typically Notch but was phenotypically facet in the heterozygous condition,

NOTCH LOCUS 755

faL/fa. Unfortunately, this mutant no longer exists; it might have been analogous to the '01 or ' lu mutants at dumpy. Mutations analogous to 'I, '01, and ' lu might actually occur at the Notch locus with a reasonable frequency. These are not detected, however, because of a bias imposed by the selection of N lethals on the basis of their dominant phenotype. In other words, recessive lethals at Notch which do not show pseudodominance of all the recessive visibles, might not have a discernible or typically notched wing phenotype when heterozygous and, therefore, never be isolated. One would predict that when lethals at this locus are selected on the basis of only their recessive lethality, the mutants analogous to '1, '01, and ' lu will then appear. It is also possible that such selection will yield complementary recessive lethals at Notch and at dumpy as well.

The discussion of the Notch locus in comparison with Ultrabithorax and dumpy suffices to indicate that this locus is not an oddity in Drosophila. One would certainly guess that in the final analysis, differences in detail will be found, but such differences will probably be due to alterations of a basic structure possessed in common by all three. The complex loci used up to now for com- parison with the Notch locus constitute a biased sample, since only those cases in Drosophila which are composed of both pseudoallelic recessive lethals and visibles have been mentioned. Before comparing and contrasting the Notch locus to other loci in Drosophila composed of only recessive visible pseudoalleles, it is best to draw some analogies with the mutants composing pseudoallelic systems of micro- organisms.

Possible analogies between mutants of microorganisms nnd macroorganisms: Since our last results at least promise to be comparable to those of similar studies performed on microorganisms, one wonders if recessive lethals in Drosophila tend to be more equivalent or analogous to the mutants ordinarily used by geneti- cists working with microorganisms (WELSHONS and VON HALLE 1960). It might be that rII mutants in phage, ad-4 or pan-2 mutants in Neurospora are more analogous to recessive lethals in Drosophila than they are to the recessive visibles. If this were so, the discontinuities observed when mapping pseudoallelic loci in Drosophila could be a consequence of the fact that the Drosophila geneticist relies heavily upon recessive visible mutants while the Neurospora or phage geneticist uses mutants which are best compared to recessive lethals in Drosophila. Thus the organisms ordinarily used by the various geneticists dictate the type of mutant choice. In Drosophila, recessive visible mutants are desirable for pseudoallelic studies, since recessive lethals, so frequently used for other purposes, are nearly impossible or extremely cumbersome in pseudoallelic work. On the other hand, a geneticist working with Neurospora, for example, will ordinarily select mutants which clearly fail to survive on minimal medium and could be descriptively characterized as recessive lethals.

One might ask, what class of mutants in Neurospora are most analogous to the recessive visibles in Drosophila? Since a number of recessive visibles can be classified as hypomorphic (MULLER 1932), i.e., mutants which characteristically have reduced enzyme activity, the class in Neurospora most comparable to the recessive visibles in Drosophila could be the leaky mutants. Hypomorphs (reces-

75 6 W. J. WELSHONS A N D E. S. VON HALLE

sive visibles) would be the mutants of choice for pseudoallelic studies in Dro- sophila, but in Neurospora the amorphs (recessive lethals) would be more desir- able.

Comparison of the Notch locus with recessive visible pseudoalleles: The dis- continuity observed when mapping recessive visible pseudoalleles in Drosophila (i.e., the localization of many alleles at a relatively few pseudoallelic sites) has been a major obstacle when attempting to make comparisons with microorgan- isms, but the analogies which have been drawn suggest an explanation. It is not difficult to imagine a broad spectrum of hypomorphic mutants at a pseudoallelic locus which have enzyme activities varying from slightly less than normal to practically nil. Depending upon the degree of hypomorphism required for the expression of a visible phenotype, a particular mutant may or may not be dis- covered. It is not hard to imagine that some of these mutants would be wild-type isoalleles, i.e., phenotypically normal when homozygous but recognizable under special environmental circumstances or various conditions of heterozygosity (STERN and SCHAEFFER 1943); in some cases, they may be detectable only by their enhancing or suppressing effects upon other quite distinct and widely sepa- rated loci (STERN and SCHAEFFER 1943). Wild-type isoalleles might therefore fill a portion of the gaps between recessive visible pseudoalleles.

There may also exist and remain undetected mutants like spl or lz’ (GREEN 1961) which differ in phenotype from the more typical pseudoalleles at a com- plex locus and which would not ordinarily be selected for study. In addition, evidence obtained from dumpy and Nctch suggests that the gaps between visible pseudoalleles might contain the mutant sites of recessive lethals in addition to the sites of postulated wild-type isoalleles and atypical visibles.

Since the burden of this paper has been to demonstrate a possible analogy be- tween recessive lethals in Drosophila and the mutants normally used in micro- organisms for pseudoallelic studies, one might expect that an enzymatic interpre- tation given to the noncomplementing mutants at the pun-2 locus of Neurospora, for example, (CASE and GILES 1960) would fit, equally well, the recessive lethals at Notch. and certainly this is the case. Thus, just as at the pan-2 locus, one can imagine a mutational change causing the formation of a protein which has been altered so that it lacks most or all of its enzymatic activity and is incapable of regaining it when in dimer or polymer association with other differentially defective proteins.

What becomes an interesting task is an attempt to interpret, enzymatically, in a somewhat similar fashion, the recessive visible pseudoalleles of Drosophila presumed to be analogous to the leaky mutants of microorganisms. Recalling that the lozenge locus (GREEN and GREEN 1956) has a limited number of separable mutant sites, and presuming that each of the genetic sites is responsible for the substitution of a moiety into an enzyme protein (GREEN 1959b), it must follow that there are relatively few positions in the protein at which an abnormal moiety substitution can yield an enzyme which in turn is responsible for the develop- mental manifestation of a more or less typical mutant phenotype. Since it has been shown that distinguishably different alleles can occur at any one genetic site

NOTCH M C U S 75 7

(GREEN and GREEN 1956; GREEN 1961) different moiety substitutions at one position in the enzyme protein might be the consequence of the different allelic mutations. Genetic changes causing moiety substitutions at other positions in the protein could yield recessive visibles that are atypical for lozenge mutants (like ZzK, GREEN 1961 ) , wild-type isoalleles, or recessive lethal mutations.

Just because the recessive visibles at a pseudoallelic locus in Drosophila might be restricted to a few separable mutant sites, it does not follow that the presumed analogous leaky mutants in microorganisms will be distributed in the same restricted way. The criterion for detection is different in Drosophila compared to Neurospora. Thus, hypomorphic mutants in Drosophila could easily be missed if they were of the nature of wild-type isoalleles or of a phenotype atypical for the locus; but presumably they might still be detected in Neurospora as a conse- quence of sluggish growth on a minimal medium.

The foregoing discussion generally implies that for pseudoallelic studies the recessive visible phenotype is a relatively imprecise end point on which to draw conclusions concerning the spatial and functional properties of a pseudoallelic locus. We wholeheartedly concur in this respect with GREEN (1961). On the other hand, if the commonly used mutants of microorganisms and recessive lethals of Drosophila are analogous, as we imply, an end point of lethality might be as precise for Drosophila as it is for Neurospora. However, a lethal in Dro- sophila might kill at any time from shortly after fertilization of the egg until the time of emergence of the adult. Thus, lethality as an end point in Drosophila could be more imprecise than in Neurospora unless the time of death, which can vary greatly, is properly noted.

POULSON (1939, 1940) showed that N s express their lethality in the egg stage. One of the N s studied by POULSON (N264-40) was utilized in our investigation although the lethal end point of all N’s in these experiments was based on the failure to observe an adult. It would be interesting to check the time of lethal expression of all these mutants and Ihe various heterozygotes as well, and it would be especially interesting to have this same information available for the ‘ I , ‘01, ‘olu and ’lu mutants at the dumpy locus.

The analogies between the mutant classes of Drosophila and microorganisms (primarily Neurospora) are speculative, and of course based on the assumptions that many more than six separable recessive lethal sites will be found to exist at the Notch locus, and that the recessive visible pseudoallelic array composed of only four mutants will eventually be shown to differ in no significant way from a recessive visible pseudoallelic array like lozenge. As noted earlier, the merit of the scheme is that it explains the apparent discordances frequently noted between pseudoallelic loci of Drosophila and microorganisms, and makes it unnecessary to postulate at this time that the organization of their genetic material is different.

Quite apart from the foregoing discussion, it should be noted that more exten- sive use of the recessive lethal in Drosophila will provide the geneticist with a relatively sensitive tool for discovering an intimate relationship between mutant sites, visible or lethal, which could not be detected otherwise. This is possible because the use of recessive lethals effectively extends the range of the non-

758 W. J. W E L S H O N S A N D E. S. VON H A L L E

complementarity phenomenon, an important tool for the investigation of gene function.

SUMMARY

It is instructive to looli at the Notch locus as if it were composed of two pseudo- allelic systems, one composed of four recessive visible pseudoalleles, the other composed of six recessive lethals. The recessive lethals appear to map continu- ously and in this way resemble the loci of microorganisms. Furthermore, the genetic interpretation of these mutants in Drosophila and microorganisms is the same.

It is suggested that the recessive lethals in Drosophila correspond to the class of mutant most similar or analogous to the mutants used by geneticists working with phage or Neurospora, and that the recessive visibles in Drosophila are prob- ably analogous to leaky mutants of Neurospora. Consequently, continuous map- ping of mutant sites as opposed to discontinuous mapping (i.e., pseudoallelic loci in Neurospora compared to Drosophila) is probably a function of the type of mutant used by the various geneticists, and the choice of a recessive visible (hypomorphic, leaky mutant) or a recessive lethal (amorphic mutant) is deter- mined to a large extent by the organisms used for investigation.

Comparisons of the Notch locus with other pseudoallelic systems in Drosophila suggest that (1 ) the Notch locus is not exceptional for Drosophila, (2) comple- mentary recessive lethals equivalent to those of Neurospora will be found in Dro- sophila when an appropriate screening technique is used, ( 3 ) the gaps between recessive visible pseudoalleles will probably contain mutable sites for recessive lethals, recessive visibles with atypical phenotypes, and possibly wild-type iso- alleles, and (4) the use of recessive lethals, which extend the range of the noncomplementation phenomenon, will enable Drosophila geneticists to discover intimate relationships between mutant sites, visible or lethal, that could not be otherwise detected.

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