COMPARATIVE MUTAGENESIS OF THE DUMPY LOCUS IN DROSOPHILA MELANOGASTER. I. X-RAY TREATMENT OF MATURE SPERM- FREQUENCY AND DISTRIBUTION1 E. A. CARLSON AND J. L. SOUTHIN2 Department of Zoology, University of California at Los Angeles, Los Angeles 24, California Received October 16, 1961 A NALYSIS of X-ray mutagenesis in Drosophila has been developed, in part, by the quantitative determination of mutation and breakage frequencies. The mutations of choice have been the sex-linked lethals because of the objectivity assured by the procedures used for their detection. The most reliable measure of breakage has been provided by the translocation test. The results from both methods permit inferences about the mechanisms of origin of these events. The frequency of lethals, for the most part, rises linearly with the administered dose and these lethals represent “simple” alterations of the gene. The translocations increase exponentially with the dose and they represent two (or more) breaks occurring independently of one another. It is also known that certain breakage events (for example, the minute deletions and inversions) also exhibit a linear relation to the dose (BELGOVSKY 1939). Furthermore, the genetic detection of such minute changes in the chromosome may be more sensitive than the cytological recognition of alterations in the salivary band patterns of the regions involved. These difficulties imply that quantitative comparisons alone are in- sufficient to determine the nature of damage inflicted by the radiation treatments ( STADLER 1954; MULLER 1956). Probably the most suitable system for a critical analysis of the events leading to mutation and breakage is the single gene. Many complex loci exist in Dro- sophila and these provide a modest degree of internal structure when tested by recombinational analysis. The problem still remains whether such complex loci or pseudoallelic systems represent a single gene or a cluster of related genes (LEWIS 1951 ; PONTECORVO 1958). Nevertheless, the proximity of these allelic sites on the pseudoallelic map is far closer than those obtained for adjacent, unrelated genes. It is also unlikely that such nests of unrelated genes will show the same mutational response as a complex locus because of the relatively inde- pendent functional roles expressed by the ‘nest’ and the complicated relations between intragenic site and functional role in the complex locus. * Portions of this work were supported by AEC Grant Contract AT 1-11 (195) to H. J. MULLER and associates, Indiana University; by a grant from the Long Island Biological Association to the senior author as Summer Investigator, 1959; and by the National Science Foundation, Grant 14222 at the University of California, Los Angeles. * Jane Shelby Jacobson Fellow in Zoology. Genetics 47: 321-336 March 1962.
Transcript
COMPARATIVE MUTAGENESIS OF THE DUMPY LOCUS IN DROSOPHILA MELANOGASTER.
I. X-RAY TREATMENT OF MATURE SPERM- FREQUENCY AND DISTRIBUTION1
E. A. CARLSON AND J. L. SOUTHIN2
Department of Zoology, University of California at Los Angeles, Los Angeles 24, California
Received October 16, 1961
A NALYSIS of X-ray mutagenesis in Drosophila has been developed, in part, by the quantitative determination of mutation and breakage frequencies. The
mutations of choice have been the sex-linked lethals because of the objectivity assured by the procedures used for their detection. The most reliable measure of breakage has been provided by the translocation test. The results from both methods permit inferences about the mechanisms of origin of these events. The frequency of lethals, for the most part, rises linearly with the administered dose and these lethals represent “simple” alterations of the gene. The translocations increase exponentially with the dose and they represent two (or more) breaks occurring independently of one another. It is also known that certain breakage events (for example, the minute deletions and inversions) also exhibit a linear relation to the dose (BELGOVSKY 1939). Furthermore, the genetic detection of such minute changes in the chromosome may be more sensitive than the cytological recognition of alterations in the salivary band patterns of the regions involved. These difficulties imply that quantitative comparisons alone are in- sufficient to determine the nature of damage inflicted by the radiation treatments ( STADLER 1954; MULLER 1956).
Probably the most suitable system for a critical analysis of the events leading to mutation and breakage is the single gene. Many complex loci exist in Dro- sophila and these provide a modest degree of internal structure when tested by recombinational analysis. The problem still remains whether such complex loci or pseudoallelic systems represent a single gene or a cluster of related genes (LEWIS 1951 ; PONTECORVO 1958). Nevertheless, the proximity of these allelic sites on the pseudoallelic map is far closer than those obtained for adjacent, unrelated genes. It is also unlikely that such nests of unrelated genes will show the same mutational response as a complex locus because of the relatively inde- pendent functional roles expressed by the ‘nest’ and the complicated relations between intragenic site and functional role in the complex locus.
* Portions of this work were supported by AEC Grant Contract AT 1-11 (195) to H. J. MULLER and associates, Indiana University; by a grant from the Long Island Biological Association to the senior author as Summer Investigator, 1959; and by the National Science Foundation, Grant 14222 at the University of California, Los Angeles.
* Jane Shelby Jacobson Fellow in Zoology.
Genetics 47: 321-336 March 1962.
322 E. A. CARLSON A N D J. L. SOUTHIN
The choice of the dumpy gene (dp-2, 13.0) for such an analysis of muta- genesis resides in such factors as the variety of phenotypic expressions, the high mutation rate and the detailed mapping which is made possible because of the fortunate location of the dumpy region on the second chromosome. The response of the dumpy region to environmental and genetic modifiers also provides an opportunity for the design of special stocks which can reveal much about its de- velopmental and physiological activity. The use of X rays for analysis of this region provides considerable information about the response of the dumpy gene to breakage resulting in loss of the entire region or in position effects exerted by the presence of new chromosomal environments. It also offers quantitative treat- ment of frequency-dose relations for the breakage events as well as for the ap- parent gene mutations. Earlier results ( CARLSON 1958a) using inseminated females treated with high and low doses of radiation did not yield sufficient in- formation for many of the problems raised. The results of the various X-rayed series presented in this paper permit an enlargement on these problems.
A general theory of mutagenesis at the dumpy locus requires a comparative approach, using many mutagens, with all the tools of genetic analysis used to their maximum powers of resolution. Because of the complex chemical and physical alterations produced by X rays in their passage through the cells, it is difficult to pinpoint with certitude the particular event which leads to a mutant phenotype. In this sense, X rays should be the last agent used for analyzing a genetic region. The ideal approach would employ agents which exclusively break chromosomes or agents which exclusively result in chemical alterations of the chromosome. Such agents are rare, if they occur at all. Nevertheless, if the genetic analysis does not clarify the mode of origin of each X-ray-induced mutant phenotype, at least it points out the limitations of present techniques and ap- proaches for a solution of these problems.
MATERIALS A N D METHODS
Detailed description of the dumpy alleles have been published earlier (CARL- SON 1958a). The main characteristics used for these mutation studies are the reduction in wing size, especially along the posterior-medial border, resulting in oblique wings (0) ; the disturbance of bristle and microchaete patterns, especially the dorso-central bristles, resulting in a vortex (v) phenotype; and the embryonic lethality (1) associated with the dumpy series. All these expressions are recessive and complement one another, expressing in the diploid whatever traits are com- mon to both dumpy genes. Genetic and environmental modifiers may sometimes be used to bring about a partial expression of these traits in the heterozygous compound containing a wild-type dumpy gene. The designations for the various alleles, which combine one or more of these traits, have arisen historically with- out a consistent nomenclature. For this reason a simplified symbolism is used throughout this article, employing those features which are most striking when the allele is in heterozygous compound with the mutant dumpy. The various systems used are presented in Table 1. The mapping of the various alleles was achieved by use of the cis-trans test (LEWIS 1951) employing the outside markers
MUTATIONS AT D U M P Y
TABLE 1
Nomenclature of the dumpy series
323
Name Old New Abbreviated of allele symbol symbol symbol
echinoid (ed--2, 11.0) and clot(cZ-2, 16.5). The former roughens the eye texture and the latter results in a dark brown eye color. Figure 1 shows the present status of the dumpy map. In all instances the recovered exceptions were mated to ed ou cZ flies to reconfirm the crossover as well as the chromosome with which it was detected. Usually two or more such crossovers were obtained in each pair of the alleles tested for their position on the map. The details of these procedures are also given at length in the 1958a paper.
There are three aspects of the mapping process which require special attention. The alleles 02 and Zu are lethal in the heterozygous condition with one another. Hence their present position on the map is not precisely known, but both are situated between the alleles cm and ou. SecoDd, the alleles expressing an (olv) phenotype, because of their extreme effects, are difficult to use for mapping with some of the viable alleles because of the reduced yield of such heterozygotes and because of their physical weakness. Partial mapping has, however, been achieved for a few of these alleles (see Figure 2) but it is too early to tell at present whether this class of mutation can be localized to more than one portion of the dumpy map. The third problem involves the degree of resolution possible in the dumpy region. The separation of the alleles oz and obm by SOUTHIN suggests that a “fine structure” map may exist. Localization of six alleles of the ou “sublocus” between Zv and u by SEDEROFF and CARLSON 1961, strongly suggests a unique association between the phenotype expressed by most of these alleles and their likely position on the dumpy map.
The series employed in these radiation studies were studied over a three-year period. The control, series 1 and series 2, employed Ore-R wild-type males which were used to inseminate ed ou cl females. The females in series 1 and 2 were X-rayed 24 hours after insemination. Series 3,4, and 5 are identical in genotype to series 1 and 2 but the males were X-rayed two days after emergence and then mated to the ed ou cl females. Series 6 and 8 employed the alleles OZV’ and Zu’, respectively, both balanced with the Curly inversion, al* Cy , ZnCyL, cnz L4 sp”. Originally these two series were intended for reverse mutation studies, with the Curly chromosome serving as a measure of the forward mutation induced at the dumpy locus. The spermatids of these two series were studied in series 7 and 9,
324 E. A. CARLSON A N D J. L. SOUTHIN
Ob" OL I" C m2 (3 ou' ut
I" cm2 fv' ov'
O2 Im cmZ Iv' ov' V L
014 ov'
c m2 0lS
Im ov'
02 0lS
0lS VZ
02 ov'
OZ V2
FIGURE 1.-In each instance the pair of alleles used was ordered with respect to one another by the placement of the ed and cl markers in the recombinant exceptions. The symbols of these alleles represent: o = oblique wings; 1 = lethality; v = thoracic vortices; cm = thoracic commas. See Table 1 for the formal designation of these alleles.
the males being freshly mated on the sixth day after treatment. The females in all these series were ed ou cl as before.
The culture conditions in all series was the same as that reported for series 1 and 2. Briefly, the flies were raised in half-pint milk bottles on standard Dro- sophila medium enriched with yeast. The number of flies per bottle was de- termined both empirically and by calculation to provide uncrowded, but not undercrowded, conditions for the emergence of the adults. Cellucotton was used
M U T A T I O N S AT D U M P Y 325
01"' VZ
Ohbm nd
olv' ov'
FIGURE 2.-The position of the alleles in these tested pairs was determined by the method described in the legend for Figure 1. Note that only olvw has been precisely localized in the map sequence presented in Figure 1 .
to provide a dry environment for some of the weaker dumpy exceptions which otherwise would have failed to survive.
RESULTS
In Table 2 the mutation frequencies are presented for the various treated series. The recovered dumpy exceptions were isolated from the nondumpy progeny and then tested for their transmissibility. Not all exceptions could be tested. The main difficulty in this study was the weakness of many of the extreme (olv) exceptions. These would often be infertile or they would die before in-
326 E. A. CARLSON A N D J. L. S O U T H I N
TABLE 2 Frequencies of dumpy exceptions induced by X rays in mature sperm
1\laiinial Minimal Nuniber ( d p j Total frequenrq- ‘Trans- frequency
* Series 1. 2 , and Control from CARLSON 1958a; series 3, 4, and 5 carried out at Iridiana University, 1958: series G, 7 , 8, and 0 carried out a t Cold Spring IIarbor, 1059.
-- Measured as the number of genetically transmitted dumpics to the total fertile survixing exceptions of ( d d phenn- t&. A gonadal mosaic is transmitted when three classes are obtained. fed iivl clj. (+ j, and (dp) . where the (dpi phenotype nsually evpresws a different allele
The males nerc iemorrd fiom the female? after 48 ho& To test speimatds. the nialei from sene5 (1 and 8 were mated to 1 irgin female5 for foui additional d a w and then separated and mated to ftmdlr, of 5eiie\ i and r)
In series 3. I.. 5 6 and 8 the males were mated n”iediate1y a f h treatment
semination, usually by becoming stuck in the food medium. Among the sur- vivors 74 out of 103 exceptions transmitted the mutant phenotype to their proge;7y in whole or in part. The average success of transmission for all the exceptions is about 72 percent. The transmission is much higher for those “comnlete” mutants which express the mutant trait in all those parts of the body which are capable of expressing it. This transmission in the complete mutations reaches 95 percent (58/61). Those mutants with mutant and nonmutant somatic tissue (“mosaic” mutants) showed a lower transmission, averaging about 38 percent ( 16/42).
Not too much difference exists between the maximum frequency of mutation and the minimum frequency because of the coincidence of a high rate of trans- mission and the high ratio of complete to mosaic type of mutations. The maxi- mum frequency is determined by the ratio of recovered dumpy exceptions to the total progeny scored whether or not they are transmissible. The minimum fre- quency is determined by taking that fraction representing transmissibility and multiplying it by the maximum frequency. It is unlikely that the bulk of the nontransmissible mutants represents phenocopies, mutations at other loci, or
MUTATIONS AT D U M P Y 327
manifestations of modifiers. The uniformity of culture conditions assures against such sporadic phenocopies and the difference between transmission of complete mutations compared to mosaic mutations suggests that a genetic mechanism is involved. Expressed as a per locus frequency, the inseminated females (series 1 and 2) average 73 X 10-*/locus/roentgen. For the mature sperm in series 3,4, and 5 it is 62.4 X 62.2 x lo-*, and 85.2 x lo-*, respectively, with an overall average for these three of 68 x 10-*/locus/roentgen. The calculations assume that the induced mutation represents a single causative event for a complete or mosaic exception. This could be accomplished by “rotational substitution” for the com- plete mutations (MULLER, CARLSON and SCHALET 1961) or by direct alteration of the single strand (such as base loss or base alteration in a DNA half-molecule) for the mosaic mutations. It might be more advisable, in the absence of direct proof for such mechanisms, to calculate these frequencies as mutations/locus/ strand/roentgen. In this case a complete mutation (affecting all the gonadal tissue as well as all the somatic tissue) would be counted as one mutant and a half-and-half mosaic as one half of a mutation. The frequencies, per strand, for these series are:
series 1 and 2 = 56.1 x 10-*/locus/strand/roentgen series 3 = 55.8 x 10-*/locus/strand/roentgen series 4 = 55.5 x 1 O-*/locus/strand/roentgen series 5 = 68.6 x 10-8/locus/strand/roentgen average 1-5 = 55.8 x 10-s/locus/strand/roentgen
These results indicate that the mutation frequency induced in inseminated fe- males at 4000r is approximately that induced in mature sperm in the male at doses of 4500 to 5000r.
The distribution of the alleles in Table 3 is based on phenotypes recovered, not phenotypes transmitted. The agreement between the two procedures is good, with the (olv) and (lv) recovered phenotypes invariably breeding true to type if transmissible. In the (ov) phenotype, however, there are two transmissible types which differ from the original phenotype. One of these, the consequence of a heterochromatic rearrangement expresses a variegated position effect ( CARLSON 1958b). Since they represent gross rearrangements they occur with expo- nentially higher frequencies at higher doses than at lower doses. Of course, such position effect mutations are not alterations of the dumpy region itself and hence should be considered as separate categories. About 20 percent of the (OV) ex- ceptions at high doses are rearrangements of this sort. The other exceptional transmission of an (ov) recovered mutant involves the occurrence of (olv) progeny instead of the expected (ov) progeny. The progeny were more extreme than the parental form. Two such instances were detected in these series. A similar transmission of an (01) phenotype to the progeny of a phenotypically ( 0 )
individual also occurred. These conceivably could be due to genetic modifiers present in isolated individuals which were not transmitted to the progeny-. They could also be due to “periclinal” mosaics, whose haphazard distribution of mutant cells in the original exceptions affected only a portion of the tissues capable of manifesting the dumpy phenotype.
328 E. A. CARLSON AND J. L. SOUTHIN
TABLE 3
Phenotype distribution of recovered dumpy alleles
Series Distribution of mutant olu
1.
2.
3.
4.
5.
6.
7.
8.
9.
TOTAL
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
Complete Mosaic Total
16 4
20
10 6
16
20 2
22
6 1 7
4 1 5
10 2
12
0 0 0
5 0 5
3 1 4
74 17 91
0 01 lu ou U cm ~-
2 0 5 3 1 0 2 3 2 I 3 0 4 3 7 4 4 0
1 2 2 1 0 0 2 2 1 3 8 0 3 4 3 4 8 0
2 1 7 6 1 0 1 0 0 6 1 0 3 1 7 12 2 0
1 0 3 1 0 0 0 0 0 2 0 0 1 0 3 3 0 0
1 0 7 2 0 0 2 0 1 5 0 0 3 0 8 7 0 0
0 0 6 1 0 0 0 0 0 0 0 0 0 0 6 1 0 0
1 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0
1 0 3 0 0 0 0 0 0 0 0 0 1 0 3 0 0 0
0 0 1 0 0 0 0 0 1 0 2 0 0 0 2 0 2 0
9 3 35 14 2 0 7 5 5 17 14 0
16 8 40 31 16 0
Total
27 15 42
16 22 38
37 10 47
11 3
14
14 9
23
17 2
19
2 0 2
9 0 9
4 4 8
137 65
202
The distribution shows that about 45 percent of all X-ray-induced dumpy mutants express the extreme (olv) phenotype and about 25 percent are (lv) in phenotype. These two classes constitute the bulk of exceptions in any given series. The other alleles are less frequent, especially the (v) phenotype which is seldom transmitted and the (cm) phenotype which was not detected in any of the radia- tion series. The (cm) expression consists of two comma-like indentations at the anterior end of the thorax associated with little or no vortex or oblique effect. ( MEYER 1955).
The mosaicism is of special interest because it accounts for 32 percent of all
MUTATIONS AT DUMPY 329
dumpy phenotypes. In virtually every instance the allelic types present in the dumpy series were recovered as both mosaic mutations and complete mutations. As previously noted, these complete mutants are usually transmitted to all of the somatic and germinal tissue of the exception; but the mosaics are apparently SO
distributed that somewhat more often than not, none of the gonad receives the mutant gene which is expressed in some of the somatic cells. Furthermore, in the remaining mosaic individuals, the gonad usually receives both mutant cells and nonmutant cells.
In addition to the dumpy mutants recovered in these nine series, the mutant clot was detected in 20 instances. Nineteen of these clot mutants were complete in phenotype and one was a mosaic affecting one eye and not the other. It might seem that more mosaics occurred than were observed, but since each fly was turned over for inspection and since the clot eye color is pronouncedly different from the wild-type red eye, it is unlikely that such a subjective error occurred. Furthermore, the number of such induced mosaic clot mutations would not be expected to be much higher, because the chance of an eye being mutant-side down is 0.5 and thus subjective misses of this sort would not have exceeded four or five if no attention was paid to turning the flies over. However, if mosaicism for eye color is more likely to be distributed in an anterior-posterior direction than is the case for dumpy (i.e., both eyes mutant or nonmutant), then this could result in a substantial number of misses. It is our opinion that this is not the case because all of the “complete” clot mutants that were tested for trans- mission were successful in passing the trait to all their progeny. The numbers are so small, however, that this problem of eye mosaicism must be explored in greater depth.
The localization of X-ray-induced mutations was carried out with the alleles listed in Table 4. These localizations indicated no differences in the approximate map sites within the dumpy region compared to those sites characteristic of spontaneous mutants with the same phenotypes. No lv mutant tested which is capable of normal recombination with ed and cl failed to demonstrate pseudo- allelic recombination as well. Similarly the olu mutants which showed no re- duction in crossing over between the ed and cl markers also showed no instance of failure to recombine pseudoallelically.
DISCUSSION
The detection of breakage in or near the dumpy region is facilitated by the occurrence of variegated position effects, deficiencies which include all or part of the dumpy region, and minute rearrangements resulting in stable position effects or in a spatial separation of the components of the dumpy region. These three types of events can be distinguished with some degree of success. Most readily diagnosed is the variegation which leads to a phenotype distinct from any other allelic expression in the dumpy region, especially in the asymmetry of wing and thorax effects, in the suppression of the mutant expression by added hetero- chromatin, and in the almost complete suppression of crossing-over in the ed-cl region. In contrast to this, small deficiencies which include the dumpy region
330 E. A. CARLSON A N D J. L. SOUTHIN
TABLE 4
Recommbination studies of X-ray-induced dumpy alleles
lllleles used Confinned recontbinants Total count
1 7* 1
6 14 3
16,947 7,100
14,230 5,806
28,270 16,795 1,151
14,455
1,795 3,835 7,675 4,597
12,428
5,547 48,971
1 14,111
7,785 26,280 22,698
* One additrnnal crossover suggested that 0 8 % was tn the right of cnP. Tl+ is the first such inr tanw of mapping
+ Spontaneous alleles used for comparison. contradiction in alnioct 200 recovered single crossovers in almost fifty painvise senei.
usually express a Minute bristle phenotype, most likely because the unrelated gene, M ( 2 ) Z (a Minutebristle mutant) is located about 0.1 map units to its left. The phenotype of such a deficiency is (olv), and hence it resembles the non- deficient o h mutations. Such small deficiencies interfere with the crossing-over between ed and dp or between dp and cl, but the reduction is not as dramatic as in the gross rearrangements leading to variegation (unless the ed marker is included in the deficiency, as in Df ( 2 ) M B ) . A deficiency will always lack any normal activity for this locus and hence these represent an amorphic type of mutation (MULLER 1932). The third class of structural changes at the dumpy locus involves minute rearrangements such as olur(dpRf), the ruffled allele of dumpy-truncate found by SCHULTZ. This mutant also reduces the amount of crossing-over between ed and cl but it does not express a Minute phenotype. Presumably the best way of distinguishing such breakage events more critically is by cytological examination. Earlier studies by MORGAN. SCHULTZ. BRIDGES and CURRY (1939) have demonstrated that the region 24D to 25A is involved in the dumpy region. Such cytological examination is limited, however, to alter- ations of not much less than one band in length, otherwise the interpretation of alteration would verge on the subjective.
Unfortunately many of the deficiency type olu mutants are killed before they breed because of their low viability and weak physical condition. This could be
MUTATIONS AT DUMPY 33 1
circumvented, if such stronger mutants are desired, by reducing the temperature in which the experimental cultures are raised to about 18°C. This would tend to partially repress the mutant phenotype; hence the more extreme olu types would survive because the ou allele with which they would be heterozygous would act considerably more in the wild-type direction at this temperature than at 28°C. The extreme deficiency types were sacrificed in these series because of the more urgent need to recover as many phenotypes as possible. The 0, U , and ou types at low temperatures would probably have been undetected or reduced in frequency because they would have overlapped the wild type in appearance. For these reasons the frequencies of cytologically visible olu mutations cannot be de- termined from the series used here.
It is appropriate to ask, however, if the remaining olu mutants represent de- ficiencies or rearrangements of a much more reduced scale, not detectable cyto- logically. These presumably would be small enough to show normal recombina- tion with the ed and cl markers. Additionally, as deletions of a small portion of the dumpy gene, they would not exhibit the Minute bristle phenotype. There are two reasons for believing that these types of ultraminute deficiencies or re- arrangements do not represent the totality of recovered olu mutations induced by the X rays in these series. First, the incidence of olu phenotypes among the mosaically arising exceptions is 17/65 or 26 percent but it is 74/137 or 54 percent for the complete olu exceptions. As will be discussed shortly, this difference suggests that the mosaic mutations are less likely to represent structural changes than the completes. Second, the results of Table 4, extending earlier work of CARLSON and SOUTHIN 1959b, show no indication that these olu mutations inter- fere with intragenic recombination. This is an important point, because if such mutants did represent a continuum of two-break events ranging from 0.1 map units to one or more map units, then a random sample of olu mutants (those showing no reduction in recombination in the ed-cl region) should include lesions of varying size up to 0.5 map units (which would escape detection with an error of 20.5 map units for normal recombination in the ed-cl region). This was not the case because the six instances of recombination tests with other alleles attempted with these olu mutants were successful, and there was no phenotypic prejudice which could indicate whether these olu mutants represented point mu- tations or ultraminute rearrangements. Similar results were found for the re- combinational properties of true minute deficiencies and for pseudoallelic point mutations in the lozenge series (GREEN and GREEN 1956). If this reasoning is correct, then the argument that X-ray point mutations represent breakage events on a submicroscopic scale would have to be abandoned, or modified with an ad hoc argument which provides for two types of breakage events. In one of these, the breaks would be so close together that the deletions or inversions would not interfere with pseudoallelic crossing-over. In the other class, the two breaks would be much further apart, involving so-called “minute rearrangements” that involve one or a very few genes. This latter type would prevent pseudoallelic recombination and also show a marked decrease in recombination in the ed-cl region. Both types would follow the “law of the linearity of frequency to dose.”
332 E. A. CARLSON A N D J. L. SOUTHIiY
The absence of a detectable intermediate size of breakage events makes such an hypothesis improbable.
If it is more likely that these pseudoallelic dumpy mutations are gene muta- tions, the mechanisms of X-ray mutagenesis must include the mosaic as well as the complete mutations. Earlier views included the concept of heterogeneously constituted semen, with some sperm containing two chromatids per chromosome and the rest with a single chromatid per chromosome (PATTERSON 1933). The idea that the chromatid might be sensitized but not fixed until after an act of replication by some sort of copying error was also considered (MULLER 1956). However, any theory of mutagenesis today must take into consideration the structure of DNA and its manner of replication. If this is done the WATSON- CRICK theory of the structure of DNA provides an immediate mechanism for the origin of mosaic mutations. The simple alteration, by X rays, of a single purine or pyrimidine base in a single strand of the duplex molecule is sufficient to account for the type of mosaic induced by X rays. The fixing of the mutant in both strands of the duplex molecule as a perpetuating part of the sequence of nucleotide pairs for the dumpy region would not take more than one or two replications as the high transmission rate indicates. It might be wondered, then, why all mosaics are not gonadally mosaic if the mutant is fixed as early as the first division. Probably the most satisfactory answer rests in the time of formation of the germ cells, which is still uncertain in Drosophila, although it may be as early as the seventh to ninth nuclear divisions, since two to seven cells, not determinate in their lineage, invade the posterior of the egg at the ninth nuclear division and form a pinched-off segment known as the polar cap. It is these cells which later invade the gonadal primordium in the sixth hour embryo. The primordium is of a separate, blastodermal origin in the embryonic development. Hence mosaicism, to a large degree, depends on such fortuitous circumstances as the migration of mutant and nonmutant-bearing cells and their subsequent morphogenetic distribution which may or may not include the induced dumpy mutation (MORGAN and BRIDGES 1919; SONNENBLICK 1950).
A paradox then arises for the origin of the complete mutations, which consti- tute such a large proportion of the mutants induced in this region. Indeed. with- out the use of an allelic series with quantitatively or qualitatively varying alleles, it would have been difficult to resist the superficial interpretation that the com- plete mutants represent deletions o r structural rearrangements of the gene. It was the compelling necessity for a complementary mechanism of mutation for the complete mutations in these radiation studies of the dumpy region which pro- vided the basis for a theory proposed and developed by MULLER, CARLSON and SCHALET 1961. In this theory, most of the complete point mutations are con- sidered to arise by a rotational movement of the base-pair, after its separation from the two linkages to the deoxyribose components of the DNA backbone. In effect. this type of “rotational substitution” constitutes an extremely minute inversion of the base-pair within the backbone. This preserves the comple- mentary relations of the mutant, satisfies the structural requirements of the WATSON-CRICK theory, and accounts for the total transmission of the complete
MUTATIONS AT DUMPY 333
mutant to its progeny. That this applies to a more general interpretation of mutagenesis than the dumpy locus itself is shown by the studies of ALTENBURG and BROWNING (1961) using genes for visible characteristics carried on alleles in the X chromosome, which indicated the high incidence of complete relative to mosaic type mutations for these loci.
One case of mosaicism is of special interest. In series I, a mutant was isolated which expressed an oblique wing ( 0 ) phenotype on one side and a truncate (olv) phenotype on the other. Both phenotypes gave rise to their own mutant line and these two alleles were designated obm and olubm because of their origination in a bilaterally mosaic individual. Preliminary analysis of these two mutants indi- cated that they were free of gross rearrangement and they were further demon- strated to recombine within the dumpy region (CARLSON and SOUTHIN 1959a). The present analysis, based on results in Table 4, shows that obm is at the extreme left end of the dumpy map, separable from os by a very low frequency. The other mutant o2vbm is also in the left end of this region, to the left of cm2, but exactly what its location is with o* or obm is still not determined because of its low viability with these alleles. The fact that these two arose together in one indi- vidual from a mature sperm treated with X rays is strong grounds for considering that two strands of the DNA were altered at different sites, probably by a single ionizing track. It is not impossible, however, that an ultraminute deletion (repre- senting obbm) was inserted in the duplicated sister chromatid after duplication in the zygote with an exertion of a position effect resulting in the ohm “allele.” Such a mechanism apparently accounts for a spontaneous bilaterally mosaic Notch (1, 3.0) and Confluens (1, 3.0) individual analyzed by SCHALET (1957). In SCHALET’S case, however, the region involved was not analyzed cytologically or by recombination analysis and its total length is unknown.
There are other aspects of the radiation mutagenesis at the dumpy locus which these studies bring to attention. A comparison of the dumpy results with those of the clot region shows a difference in the ratio of apparent complete to mosaic mutations. Whereas the dumpy region expressed 34 percent of its mutations as mosaics (65/202), the clot region expressed only five percent ( 1 /20). Assuming that the sampling error in the clot region does not make this an unduly small percentage and that the mosaicism is bilateral and not primarily of an anterior- posterior distribution, this would imply that a difference exists in the relative amount of true gene mutation occurring in these two regions. Inspection of the dumpy data points out that a higher percentage of olv mutants exists as completes than as mosaics in contrast to the distribution of complete to mosaic mutations for all other classes of alleles. Perhaps the excess of olv types in the complete muta- tions represents mainly the minute deficiencies and rearrangements produced by breakage in this area. This would amount to about 25 to 30 olv exceptions of the deficiency or rearrangement type among the 74 complete olv exceptions induced. The exact number is difficult to ascertain because of some anomalous differences between mosaic and complete mutation and transmission frequencies in the other alleles, which make these calculations somewhat tenuous. Nevertheless, enough viable structural alterations of the olv type were recognized to suggest that a
334 E. A. CARLSON AND J. L. S O U T H I N
figure of this magnitude (between one-fifth and one-half of the complete olu mutants) is reasonable (CARLSON 1958a).
If breakage in the clot region is not appreciably different from breakage in the dumpy region, a similar number of such detectable structural alterations should exist for it. Thus the clot region would be expected, on this interpretation, to have produced no significantly different number of structural alterations leading to a clot phenotype than were produced in the dumpy region leading to the breakage classes of olv mutants. However, if the true gene mutation fre- quency of the clot region is considerably lower than that of the dumpy region, then the production of gene mutations (mosaic or complete) would be small. And if the same ratio of about one-third to one-half holds for the incidence of induced mosaic gene mutations at the clot locus to the total of clot gene mutations, then the one or two complete point mutations predicted in excess of the single mosaic obtained would be swamped by the relative abundance of breakage events in this region. It is this type of difference between true gene mutation frequency at different loci in the presence of a relatively constant breakage frequency in the area of each locus, that may account for the impression that breakage rather than gene mutation is the major mechanism involved in X-ray mutagenesis ( STADLER 1954; FRYE 1961). The true gene mutation difference existing between clot and dumpy could be a reflection of either size (with dumpy about 20 to 50 times larger by these calculations) or a difference in response of the altered gene to its normal function. In this latter situation, a gene may be relatively refractive to mutation if substitutional alterations of parts of the molecule do not lead to a functional change in its enzymatic or structural product. However, some genes might be very sensitive to all alterations of the gene molecule. expressing a marked change in function of the gene product and hence a mutant phenotype. These speculations require biochemical and physiological levels of experimenta- tion which are not yet feasible in Drosophila, but which, in part, seem to exist in viral systems (BRENNER, BARNETT, CRICK and ORGEL 1961). Unfortunately these clot mutations were not saved as stocks for cytogenetic analysis, but the point seems sufficiently important to justify a re-examination of this problem with the clot region or with other genic systems similar to it but more amenable to cytogenetic analysis.
The attempt to obtain reverse mutations was temporarily abandoned when none appeared with X-ray treatment in three series used (series 6 and 7, 8 and 9, and homozygous ou') . The totals in these three series for the dumpy-bearing chromosomes are 4045, 8490, and 15,140, respectively. With the use of a selective system this problem of reverse mutation should be more critically examined (CARLSON and SEDEROFF 1961). However, in series 8 and 9, the 10 allele did not prevent the recognition collectively of induced olu, 01, ov and o mutations. These amounted to 18/8490 (0.21 % ; 16 complete and two mosaic). In the various high- dose series in which nondumpy chromosomes were irradiated, a total of 121 mutations of these four classes with an o effect was detected in 55,092 progeny (0.22% ) . There is no difference between these two values and the presence of the lv allele in the chromosome does not appear to alter the induced rate of mutation
MUTATIONS AT DUMPY 335
at other sites in the dumpy region. Studies using mutational isoalleles differ somewhat from this approach since phenotypically similar alleles of a wild-type locus are found to respond with varying degrees of difference to X-ray and spon- taneous mutagenesis (LEFEVRE and FARNSWORTH 1954). Other mutant loci in the dumpy series need to be tested for their effect on forward mutation before any meaningful interpretation can be made on how these systems compare.
A comparison of the X-ray results with those obtained from treatments with monofunctional quinacrine mustard will be discussed in a separate article (CARL- SON and OSTER 1962).
SUMMARY
1. Evidence is presented to support the view that the majority of X-ray- induced mutations at the dumpy locus are gene mutations not associated with multibreakage events.
2. Nonvariegated mosaicism at the dumpy locus is usually free of detectable breakage events. Complete mutations (those affecting the entire body) form a mixed class of breakage events and gene mutations.
3. The mutation frequency of the dumpy gene was not altered when sperm bearing a pre-existing dumpy mutation were used for irradiation.
4. Gross mutation detected at the clot locus is one tenth that of the dumpy locus. Criteria are evaluated for determining the true gene mutation frequencies at both loci.
5. The results of the X-ray analysis of the dumpy locus support a theory of mutagenesis proposed by MULLER, CARLSON and SCHALET ( 1961 ) .
ACKNOWLEDGMENTS
We are indebted to PROFESSOR H. J. MULLER for his penetrating criticisms of this paper and for his encouragement throughout its progress.
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