analysis of dysgenesis-induced lethal mutations on … · copyright q 1984 by the genetics society...

Copyright Q 1984 by the Genetics Society of America

ANALYSIS OF DYSGENESIS-INDUCED LETHAL MUTATIONS ON T H E X CHROMOSOME OF A Q STRAIN

OF DROSOPHILA MELANOGASTER

MICHAEL J. SIMMONS, JOHN D. RAYMOND, TIMOTHY P. CULBERT AND TODD R. LAVERTY

Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108-1095

Manuscript received August 12, 1983 Revised copy accepted January 6, 1984

ABSTRACT

The Q strain known as U6 was tested for its ability to induce X-linked lethal mutations in male and female hybrids from crosses with M strains in the P-M system of hybrid dysgenesis. All measurements of the'mutation rate were made on the X chromosome derived from the U6 strain. The lethal rate for young hybrid males from the cross M female X U6 male was 1.1 1 % per chromosome. For older males, it was only 0.4495, suggesting that there is less mutational or more repair activity in the germ cells of the older males or that mutant cells are selectively eliminated as the hybrid males age. The lethal rate for hybrid females from comparable crosses was approximately the same for both ages that were tested. However, it was substantially less than the rate for the hybrid males-only 0.26% per chromosome. Genetically identical hybrid females from reciprocal crosses also showed a low mutation rate, 0.13% per chromosome. Again, there was no difference between young and old flies.-Mapping experiments established that most of the lethal mutations that were recovered from the male and female hybrids were located in two regions on the X chromosome, one between bands 14B13 and 15A9, the other between bands 19A1 and 20A, which encompasses the maroonlike locus. More refined mapping of the lethals in the maroonlike region demonstrated that the vast majority of these affected a single gene located in band 19C4. Cytological analysis of the lethal chromosomes revealed that several carried rearrangements, including inversions, duplications and deficiencies. Chromosome breakage occurred primarily in bands 14D1-3 and 18F-20A, and most of the breaks in the latter segment were located in 19C. However, rearrangements involving 19C and mutations of the gene in 19C4 were mutually exclusive events. In situ hybridization of a P element probe to the chromosomes of & demonstrated that P elements reside at a minimum of five sites on the X chromosome. These P element sites correspond to the mutational and breakage hot spots on that chromosome. The combined genetic and cytological data imply that most of the X-linked lethal mutations that occur in M X hybrids are due to local P element action. Consideration of these and other data suggest that is a weak P strain in the P-M system of hybrid dysgenesis and that other Q strains might also be regarded in this way.

HE condition known as hybrid dysgenesis occurs in Drosophila melanogaster T when members of the P family of transposable elements are activated. This happens when the elements are introduced into a cellular state called the

Genetics 107: 49-63 May, 1984.

50 M. J. SIMMONS E T AL.

cytotype (ENGELS 1979a), which is a property of the M strains of the species. Most of the A4 strains that have been examined to date lack any trace of P elements; in contrast, strains designated as P possess many copies of these elements (BINGHAM, KIDWELL and RUBIN 1982). These strains also possess a cellular state called the P cytotype which represses P element activity.

Recent sequencing studies (O’HARE and RUBIN 1983) have established the structure of several P elements. Some are 2907 base pairs long and have 31 base pair-inverted repeats at their ends. These large elements contain three long open reading frames and are thought to encode at least two proteins. One is postulated to be a transposase that activates P elements; the other is postulated to be a regulator that brings about the P cytotype. Although neither of these proteins has yet been identified, functional studies have pointed to their existence (SPRADLING and RUBIN 1982; ENGLES 1983).

P strains also possess numerous smaller elements that are derived from the large ones by internal deletions. These deletions are thought to abolish the production of one or both of the postulated proteins. Significantly all of the small elements that have been studied to date do have the same 31 base pair- inverted repeats at their termini.

In the M cytotype P elements bring about the syndrome of germ line ab- normalities called hybrid dysgenesis (BREGLIANO and KIDWELL 1983). The term “hybrid” is used because P elements are most easily introduced into the M cytotype by hybridizing P and M strains. Since the cytotype of a fly is strongly influenced by that of its mother (ENGELS 1979a), the hybridization is usually performed by crossing M females with P males. The reciprocal cross is not expected to bring about the desired combination of P elements and M cytotype. Once activated, P elements can cause mutations and chromosome rearrangements; they can also induce a particular form of sterility called gonadal dysgenesis in the hybrid progeny of a dysgenic cross. This sterility is characterized by a failure of the gonads to develop and probably comes about by massive cell death in the germ line.

Some strains that harbor P elements produce all of the manifestations of dysgenesis except sterility whenever they are crossed with females from an M strain. Such strains have been designated as Q in the P-M system of hybrid dysgenesis (KIDWELL 1979). Recent surveys (KIDWELL 1983) of natural populations indicate that Q strains are in the majority in Europe, Africa and the Mideast, whereas P strains predominate in North and South America; M strains seem to be common in Australia and the Far East, but the data from these regions are still too limited to draw a firm conclusion. One island population off the coast of Japan is clearly Q (OHISHI, TAKANASHI and CHIGUSA 1982).

Here we report the results of genetic studies with a Q strain derived from a natural population in the United States. We collected X-linked lethal mutations from hybrids that were made by crossing this strain with standard M strains. We estimated mutation rates for these lethals and also studied their distribution along the chromosome. In addition, we examined the lethal chromosomes cytologically in order to ascertain the extent to which the P elements of the Q strain induce chromosome breakage.

DYSGENESIS-INDUCED LETHALS 51

MATERIALS AND METHODS

Strains: Q X M hybrids were made using a subline of the inbred Q strain known as v6 (SIMMONS ~t al. 1980; ENGELS and PRFSTON 1981a), which is homozygous for an autosomal recessive mutation that disrupts the venation of the wings. The M strains that were used in making hybrids included:

1. C(I)DX, y f / Y / y cin w f s~(f)"~~9, an attached-X strain described by SIMMONS et al. (1 980). 2. FM7/sr71, an X chromosome balancer strain also described by SIMMONS et al. (1980). 3. Basc, an X chromosome balancer strain possessing the Canton S wild-type background.

All of the aforementioned strains were tested by us and found to be inducers in the I-R system of hybrid dysgenesis (BREGLIANO and KIDWELL 1983).

To map the lethal mutations detected in these experiments, the stocks listed in Table 1 were used. The first five of these contained deficiencies on the X chromosome that were complemented by duplications on the Y or the second chromosome. The duplications and deficiencies permitted us to screen different regions of the X for the presence of lethal mutations, including segments around the loci U! (white eyes), ct (cut wings), in (miniature wings), r (rudimentary wings) and mal (maroonlike eyes). The lethals that mapped in the mal region were localized more precisely by using the last three stocks listed in Table 1 and a set of 11 "point" mutations lying in the 19A4- E2 interval of the polytene chromosome map. One of these was provided by D. ISH-HOROWICZ; the others came from G. LEFEVRE.

The preliminary crosses in the mapping experiments and the crosses designed to produce lethal heterozygotes for cytological examination of the polytene chromosomes required additional stocks. These were:

4. FM6, l ( l )6" /DB/~~sY (OB is Df(l)basc, a balancer chromosome with a small deficiency that acts as a recessive lethal (see SIMMONS and LIM 1980). 5. FM6, l(1)6"/Df(l)icr'/'/~wBSw+y+Y (see SIMMONS and LIM 1980). 6. y snSu (see RAYMOND and SIMMONS 1981). Most of the chromosomes and markers used in these experiments are fully described in LIN-

DSLEY and GRELL (1968). X-linked lethal tests: Hybrid males were produced by crossing vg males with attached-X females

at 25". These F1 hybrid males were then mated individually with FM7/sc7 1 females to obtain +/FM7 daughters for the X-linked lethal tests. The procedures were the same as those of SIMMONS et al. (1980).

flies at 25" with flies from either of the two balancer strains, F M 7 i k 7 1 or Basc. When v6 flies were used as the male parent, the mating was designated cross A. When they were used as the female parent, it was denoted cross B. These designations follow the conventions established by KIDWELL, KIDWELL and SVED (1 977). Balancer/ + FI daughters were selected from these crosses and mated individually at 25" to males from the corresponding balancer stock. Here "Balancer" is either the FM7 or Basc X chromosome, and "+" is the X chromosome from the strain. If wild-type sons emerged in the Fz, then the + chromosome in the gamete that gave rise to the F1 female was lethal-free; it was, therefore, possible to test for the occurrence of lethal mutations in that female's germ cells. To do this test, virgin Balancer/+ FP females were collected from these "lethal-free" cultures and mated to their Balancer/ Y brothers. Then each FP female was placed separated in a culture tube and incubated according to the procedures of SIMMONS et al. (1980) for the X-linked lethal test. The absence of wild-type males in the Fs indicated that an X-linked lethal might have been carried by the F2 female. This lethal would have occurred in a germ cell of the F1 female. All such cases were subjected to further tests to verify the presence of the suspected lethal. The classification methods of SIMMONS ~t nl. (1980) were used.

Lethal mutations that were identified in these experiments were kept in balanced stocks at 21" until they could be analyzed further. This was between 20 and 30 generations after the X-linked lethal test. In these stocks the cytotype was not controlled.

Mapping: We attempted to map most of the lethal mutations that were detected in the experiments. For the male-induced lethals, the procedure was to cross FM7/1 females to males from each of the first five duplication/deficiency stocks listed in Table 1. The cultures were incubated at 25" and scored for the presence of the deficiency/l and I/duplication classes. For most of the female- induced lethals the procedure was to cross single Balancer/l females to DB/sc'Y males, giving DB/

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1 F1 females which were then crossed to males from the duplication/deficiency stocks. The progeny of this last mating were scored for the indicator classes.

A few of the female-induced lethals were not viable in combination with the DE chromosome, which carriers a deficiency near the left end. For these the mapping procedure was like that of the male-induced lethals; Balancer/l carriers were crossed to males from the duplication/deficiency stocks, and the progeny were scored for the deficiency/l and I/duplication classes.

More refined mapping was carried out for the male- and female-induced lethals that affected the mol region. The procedure was to cross l/y+Y mallM males (providing these were viable and fertile) to females from each of the tester stocks. The mal region deficiency testers were used first; then, single gene testers were used to pinpoint the lethal on the map.

Cytological annlysis: The lethal X chromosomes recovered from the hybrid flies were examined cytologically for rearrangements. For the female-induced lethals that were viable with the DE chromosome, DEI1 F1 females from the mapping crosses were mated to y sn’v males to produce l/? sn’u larvae. These have black mouth parts and are distinguishable from their DE/y sn’v sisters, which have yellow-brown mouth parts. The salivary glands of the l /y sn’ v larvae were dissected, stained, and squashed in the usual way. The female-induced lethals that were not viable with the DE chromosome were handled differently. All of these were balanced with the East chromosome; therefore, Ensc/l carriers were mated individually with Df(l)wd’/pw+y+Y males to produce DJll)wd’/ 1 larvae, which have colored malpighian tubules; the malpighian tubules of their Df(I)wd’/Easc sisters are colorless. Thus, the larvae with the colored malpighian tubules were selected to make polytene chromosome squashes.

For the male-induced lethals, the cytological analysis was carried out prior to the mapping experiments. The procedure was to cross FM7/1 females to D E / s c ~ males to obtain DE/1 carriers, which were then crossed to p sn’u males. Female larvae with black mouth parts were selected from the progeny of this cross and dissected to obtain salivary glands for polytene chromosome squashes.

In situ hybridization: Salivary glands of third instar female larvae from two independently main- tained sublines of the v6 stock were dissected in Ringer’s solution, fixed in ethanol and acetic acid and squashed on gelatinized slides. The slides were hybridized with a tritium-labeled RNA probe made from the plasmid pzr25.1. This plasmid contains a complete P element and flanking DNA sequences cloned from the 17C2-3 region of the X chromosome of a P strain (O’HARE and RUBIN 1983). The in situ hybridizations were performed according to the procedures of BINGHAM, LEVIS and RUBIN (1981).

iMedin: A standard cornmeal-molasses medium was used in all cultures except those of the X- linked lethal tests, in which a sugar-yeast medium was used.

RESULTS

X-litiked lethal mutation rates: We monitored the occurrence of X-linked lethal mutations in the germ cells of hybrid males and females made by crossing M strains with the Q strain known as V 6 . Dysgenic male hybrids from a single cross and female hybrids from four different crosses were tested for the production of lethals. Two different M strains were used to generate the female hybrids; for each of these, reciprocal crosses were performed, giving both A and B hybrid daughters, which were genetically identical. For both the male and female hybrids, the X chromosome that was screened for the lethal mutations always came from the & strain. The data from all of the experiments are presented in Table 2.

Altogether, more than 1000 hybrid males were tested for the production of lethal-bearing sperm. The majority of these were young (2 days posteclosion) at the time of mating and came from two sets of crosses conducted under identical conditions. The rest were 9 days posteclosion and came from only one of the two sets of crosses. The X-linked lethal mutation rate for the young

54 M. J. SIMMONS ET AL.

TABLE 2

X-linked lethal inutatioii rates f o r male andfiinale hybrids from crosses between v6 and certain M strains

No. Age No. flies chromosomes No.

Hybrid (days) tested tested lethals Mutation rate * SE Pooled rate

Male A 2 706 4524 58" 0.0121 f 0.0020 Male A 2 244 2504 26b 0.0098 2 0.0022 0.0111 f 0.0015

Male A 9 196 1834 8 0.0044 f 0.0016 0.0044 f 0.0016

FM7/+ female A 2 382 1475 3 0.0020 f 0.0012 FM7/+ female A 9 376 1512 5' 0.0033 f 0.0027 Box/+ female A 2 348 2301 6 0.0026 f 0.0011 Enscl+ female A 9 383 2870 15' 0.0052 & 0.0027 0.0026 2 0.0007

FM7/+ female B 2 435 1590 3' 0.0019 f 0.0019 FM7/+ female B 9 427 1796 0 Basc/+ female B 2 349 2291 0 Ensf/+ female B 9 365 2484 13' 0.0052 f 0.0030 0.0013 k 0.0009

For the female hybrids, cross A is M female X v.5 male, whereas cross B is the reciprocal. Mutation rates were calculated according to the unweighted method of ENGEU (1979~). The pooled rates were obtained by weighting each component by the reciprocal of its variance, except for the B hybrid females, where the rate was recalculated from all the data.

Seven clusters of two. ' Four clusters of two. 'One cluster of four.

One cluster of seven. ' One cluster of three. 'One cluster of 12.

males is clearly greater than that for the old ones, corroborating previous results (SIMMONS et al. 1980). The average of the rates given here for the two experiments involving the young males is 1.11 2 0.15% whereas for the old males the rate is 0.44 f 0.16%. These values are significantly different (P < 0.05).

In contrast, the young and old hybrid females from the A crosses had similar X-linked lethal mutation rates. The average rate, pooled over ages and strains, is 0.26 k 0.07%, which is significantly lower than the rate for the young hybrid males, but not for the old ones. Evidently, hybrid females from the cross M female X Q male have a lower X-linked lethal mutation rate than hybrid males from the same kind of cross. This may be due to differences in the M strains used to obtain the male and female hybrids or to a b o n a j d e reduction in the mutational activity of the elements responsible for dysgenesis in hybrid females. KIDWELL, KIDWELL and IVES (1977) reported a high rate of mutation for female hybrids from crosses of M females X P males.

As expected, the B hybrid females have a mutation rate that is even lower than that of the A hybrid females. In two of the experiments with the B females, no lethal mutations were detected. In the other two, a total of 16 lethals were identified among the progeny of 800 hybrids. All but one of these


occurred in clusters. When ages and strains are pooled, the lethal mutation rate for the B hybrid females is estimated to be 0.13 f 0.09%, which is not significantly different from the rate for the A hybrid females. However, this rate may be overestimated because some of the lethals detected in the progeny of the B hybrids may have arisen in the V 6 stock itself rather than in the hybrid flies. This is so because the B hybrids that produced the lethal clusters segre- gated only lethal chromosomes in the sample of chromosomes obtained from them. This suggests that these hybrids were actually carriers of a lethal mutation that had been transmitted by their V 6 mothers and that had slipped through the F1 carrier screen. If this is the case, the actual rate of mutation for the B hybrid females is estimated to be 1/8146 = 0.01%, a value much lower than the estimate for the A hybrids.

Genetic and cytological analysis of the lethal X chromosomes: We performed complementation tests to localize the lethal mutations on the chromosomes derived from the hybrid flies. These involved a set of well-distributed duplications and deficiencies that covered approximately 20% of the euchromatic length of the X . Altogether, 77 male-derived lethal chromosomes were screened with the duplication/deficiency stocks. The mutations on 12 of these could not be localized to any of the five map regions that were tested. However, 41 (53.2%) of the chromosomes had mutations that failed to complement Df(l)ma13, plac- ing the lethal in the mal region between 19A1 and 20A on the polytene chromosome map. Of these 41, one also contained a lethal that did not complement Dfl)m259-4, the deficiency that uncovers the m region. The other 40 did not have second site lethals, since, for each, the lethal in the mal region was covered by the duplication. Twenty-three (29.5%) of all of the chromosomes tested had lethals that were uncovered by Dj(l)r+75c. Thus, these lethals mapped near the r locus between bands 14B13 and 15A9. One of these chromosomes had an additional lethal that was not complemented by T(l;2)r+75'; therefore, this lethal must have been located outside the rudimentary region. One last chromosome had a single lethal in the m region uncovered by Df1)m259-4. Thus, the rudimentary and maroonlike regions are mutational hot spots on the X chromosomes of V 6 male hybrids.

The data just given include pairs of lethals that came from the same hybrid male. There were nine such pairs altogether, but the members of only two of these mapped in the same location. Therefore, the preponderance of the clusters obtained from the male hybrids was composed of lethals that occurred independently. The absence of an appreciable number of double lethals is also noteworthy. It indicates that very few secondary mutations occurred during the time that the lethal chromosomes were kept in stock.

Further mapping of the mal region lethals was accomplished by using the inn1 deficiencies listed in Table 1 and a set of 11 point mutations lying in the 19A4-E2 interval on the map. Thirty-three of the 35 male-derived lethals that were tested failed to complement the lethal mutation HF326, which maps in band 19C4. However, 32 of these did complement the point lethals DC711 and DA588, which lie in bands 19C2 and 19C5, respectively. Since no lethal is known for band 19C3 (LEFEVRE 1981), the vast majority of the mal region


lethals seem to affect a single essential gene in band 19C4. One of the 33 lethals that failed to complement HF326 also failed to complement DA588 and EF489 (in band 19C6), indicating that it was associated with a small deficiency; however, this deficiency was not seen cytologically. Significantly, the left end of this deficiency involved the 19C4 mutational hot spot. The two remaining lethals among the 35 tested mapped in the 19A2-Dl interval on the chromosome. They failed to complement each other but did complement all of the point testers that were used. Evidently, these two lethals defined another essential locus in the region.

The same chromosomes that were tested for the location of the lethals were examined cytologically. Fourteen of the 77 chromosomes had rearrangements, all involving a break in the 14C8-D3 bands (see Table 3). None of these 14 rearranged chromosomes was derived from the same hybrid male. The rearrangements included inversions and duplications. Ten of the breaks in the 14C-D bands were associated with lethal mutations uncovered by the r+’& deficiency. This suggests that these breaks were the cause of the lethal effect. There were 11 breaks in the region between 19A and 19E; the precise location of these was difficult to determine but most seemed to involve 19C, and none was associated with a lethal effect. Other breaks were detected in 1A (nonlethal), 2B (nonlethal), 11A (two breaks, both nonlethal) and 18F (two breaks, one lethal, the other with an unknown effect on viability), Clearly, the sites that were most frequently broken on the U6 X chromosome lie in the regions that most often gave rise to lethal mutations.

The lethals derived from the female hybrids were also mapped; however, only two of the duplication/deficiency stocks were used, these being the ones involving the r and innl regions. This less thorough approach was adopted because by this time we already knew that the r and mal regions were mutational hot spots for the male-derived lethals.

Altogether, 25 lethals from the A hybrid females were tested, but some of these were derived from the same fly. For instance, six of seven lethals recovered from one Buscl+ A hybrid female were inviable over the DB chromosome, which has a deficiency near the left end; thus, these lethals mapped near the yellow (y) locus. The seventh lethal in the cluster mapped elsewhere. Such lethal clusters make a full presentation of the data unwieldy. Therefore, after pooling the Bnsc and FM7 experiments, we have tallied the minimum number of independent mutations. The results are given in Table 4, which also includes a tally of the 14 lethals from the B hybrid females that were tested. It is clear that the r and inn1 regions are again hot spots for dysgenesis- induced mutations; 25% of the independent lethals from the A hybrid females mapped in the r region, and 35% mapped in the mal region. For the B hybrid lethals, three of the five independent lethals were located in either of these two regions.

Detailed mapping of five of the innl region lethals from the A hybrids established that all five affected the locus in 19C4. One also affected the locus in 19C5 but was not associated with any cytologically recognizable deficiency. Another of these five lethals failed to complement all of the tester alleles


TABLE 3

Rearrangements detected among the lethal chromosomes that were examined cytologically

Male-derived lethal chromosomes Femalederived lethal chromosomes

Lethal Lethal location Aberration location Aberration

ma1 mal ?

6t(1)14D1-2;19A-D y "Df(1) tip;lAd-Bl (6 chromosomes) It1(1)14D2-3;19C y " D f l ) tip;lA8-B1 + Dp(1;1)19C;19F 1~1(1)14D1-2;19C2-5 r In(1)14C7-D1;19C2-D1 In(1)14Dl-2;19C-E r In(1)14C7-D1;19C2-D1 In(l)l4D; 19C mal Df1)192-3;19D2-E1 In(1)14D; 19C mal Dfl)l9A4-5;19F3-ZOA In(1)14D; 19C mal Df(l)l9A2-3;19DZ-E1

Dp(1;1)14Dl-2;11A8-9 into 19C + possible inversion of 14D3;19C

bDp(l; 1)14DZ-E1;18F into lA l -8

I1~~(1)2B~14Dl-2+14D~19Dl-2

h1(1)14D2-E1;18F2-5 I~ t ( l ) l lA2-6~1401-2 61(1)14C8-D2;19C2-6

The lethal locations r and mal refer to the map regions defined in Table 1; the y (yellow body)

" From a cluster. region is at the tip of the X chromosome and is defined by the deletion in Df(1)Basc.

This rearrangement is slightly viable in males. The viability reduction is probably due to the large duplication.

TABLE 4

Distributiott of indepeudent lethal mutations recovered from Q X M hybrid females

Map region Minimum no. independent

Type of hybrid Y" r b mal' Id mutations

A 2 5 7 6 20 B 0 2 1 2 5

See text for details. " Uncovered by Df(1)Basc.

Uncovered by D f ( l ) ~ + ~ ~ . ' Uncovered by Df(1)mal'.

Could not be localized.

between 19C4 and 19E2 but did complement those to the left of 19C4. This lethal was clearly associated with a deletion. Cytological examination of the chromosome that carried it indicated that the proximal breakpoint of the deletion was in 19F3-20A, and the distal breakpoint was in 19A4-5. Since there are essential loci between 19A5 and 19C4 that were not mutant on this chromosome, the results of the genetic mapping and the cytological analysis were inconsistent. This discrepancy could be due to a secondary deletion that occurred in the crosses that produced the larvae for cytology or to a poly- morphism for two different deficiencies in the lethal stock itself.


There were two other inconsistencies between the genetic mapping of these mol region lethals and the cytological data. Two of the lethals that were located in 19C4 by genetic tests were shown by cytological analysis to be associated with large deficiencies in section 19 of the chromosome. Both of these had breakpoints in 19A2-3 and 19D2-E 1 . Since these deficiencies removed several essential loci that were screened in the genetic tests but that were not shown to be mutant, they might have occurred in the crosses that produced the larvae for cytology or were segregating in the lethal stocks and escaped detection by chance.

Five other rearrangements were identified on lethal X chromosomes derived from the A hybrid females. These, as well as the rearrangements discussed earlier, are listed in Table 3. The breakpoints included sites in l A , 14C-D, 19A, 19C-D, 19D-E and 19F-20A. N o rearrangements were detected on any of the lethal chromosomes recovered from the B hybrid females.

The lethal mutations from the B hybrid females that mapped in the mal region all came from one cluster; therefore, they probably arose from a single mutational event. All nine of these lethals complemented the mal deficiency testers, except for Df(l)ma13; thus, they map near the boundaries of the mal region, either in 19A1-2 or in 20A.

In situ hjbridizations: The X chromosomes of three larvae from each of two sublines of were examined for P elements by labeling with a tritiated RNA probe made from a complete P element. One of the two sublines was the strain used to generate mutations in these experiments; the other had been main- tained independently for 4 yr in the laboratory of W. R. ENGELS.

The X chromosomes of the two sublines were labeled by the probe at the same sites: l A , 11A (distal to the constriction), 14C-D, 17C and 19A-D. The 17C site was labeled because it was homologous to non-P sequences contained in the probe. All of the other sites were labeled because they contained P elements. In well-stretched X chromosomes, the 14C-D site was labeled in 14D only, and the 19A-D site was resolved into two sites, one in 19A-B and the other in 19C-D. Thus, the X chromosomes of the larvae from these two sublines carried P elements at a minimum of five sites: 1 A, 1 l A , 14D, 19A-B and 19C-D. The number of sites is a minimum because very small P elements would probably not be detected by this technique. The results of the in situ hybridizations with ENGELS’ subline of the U6 strain confirm ENGELS’ unpublished findings (personal communication). Since both sublines examined had the same X-linked P element sites, it appears that these have been conserved during the time that the two sublines have been separated.

DISCUSSION

Mutatioiz rates: SIMMONS et al. (1980) reported that hybrid males obtained by crossing V6 males with attached-X females from the same A4 strain used here had an X-linked lethal mutation rate that was significantly greater than that for males from the U6 stock itself. They also noted that males from the reciprocal cross had a mutation rate on a par with that of the U6 males. Therefore, the enhanced mutability was seen only in males from the dysgenic or A cross.


Moreover, the mutation rate of young hybrid males (1.73 f 0.18%) from this cross was greater than that of their older brothers (0.93 f 0.14%).

The results of the experiments reported here confirm and extend these findings. The rate of X-linked lethals in the young hybrid males from the A cross is estimated to be 1.11 f 0.15%. When the result of the previous ex- periment is included, this estimate rises to 1.36 f 0.12%. The combined mutation rate for the older males is about half this value, suggesting that there is less mutational activity in their germ cells or that there is more repair of mutational lesions. Another possibility is that mutant germ cells are selectively eliminated as the males age.

Genetic analysis of the lethals that occurred in the A hybrid males indicates that these mutations are located primarily in two small regions of the X chromosome. One region, around the rudimentary locus, consists of 3-4% of the euchromatic portion of the X, the other region, around the maroonlike locus, also consists of 3-4% of the X. Together these two regions account for more than 80% of the lethals that occurred in the dysgenic hybrid males. In absolute terms the remaining 20% amounts to 0.22% lethal chromosomes per generation, which is approximately equal to the total lethal rate for nonhybrid v6

males. Therefore, this residue of lethals is probably due to events unconnected with dysgenesis.

In hybrid females from 4 X M crosses, the X-linked lethal rate is lower than it is in hybrid males. For females from the A cross, the rate is 0.25 f 0.07%; for those from the B cross, it is 0.13 & 0.09%. In neither case is there a detectable age effect. The few lethals that do occur in the hybrid females tend to map in the rudimentary and maroonlike regions, indicating that, although the mutation process is itself depressed, it acts in the same chromosomal regions that are mutated in hybrid males. This suggests that the mutator mech- anism is the same in both sexes but much less effective in females. One possible explanation is that the enzymes responsible for recombination manage to repair many of the mutations caused by dysgenesis in females. Since these enzymes do not function in males, the male mutation rate is higher. Another possibility is the explanation given by ENGELS (1981) for the greater instability of a dysgenesis-induced mutation of the singed locus: The germ cells of males go through more cell generations than females; therefore, they might accumulate more mutations if the rate per cell generation is constant.

Mutational and breakage hot spots on the V 6 X chromosome: As mentioned, two segments of the V 6 X chromosome contain mutational hot spots for hybrid dysgenesis. The segment we have called the rudimentary region extends from bands 14B13 to 15A9 on the polytene chromosome map; the other segment, called the maroonlike region, extends from 19A1 to 20A. A more refined analysis of the mutations located in the maroonlike region shows that the vast majority of these affect a single gene located in band 19C4. This locus is, therefore, a preferred site for dysgenic mutator activity on the v6 X chromosome. In a few cases, it seems to be the endpoint of dysgenesis-induced deletions.

The cytological analysis of the lethal chromosomes obtained from the V 6


hybrids reveals that at least two sections of the V 6 X chromosome have a propensity to break under the influence of dysgenesis.. These are in the bands 14D1-3 and 18F-20A, situated in the rudimentary and maroonlike regions, respectively. Clearly, the sites with the greatest number of breaks correspond to those with the greatest number of mutants. However, it is not clear from the cytological analysis how many breakage points actually reside in each of these segments of the chromosome. The 14D1-3 bands may have two breakage points, since both lethal and nonlethal breaks were detected. Another possibility is that the 14D1-3 bands contain a single main breakage site that is not lethal, but that sometimes a break at that location is accompanied by a small deficiency that has a lethal effect.

The situation in the 18F-20A region may be even more complex. From our analysis it was not possible to determine the precise location of all of the breaks in this region, but several involved the 19C section. The combined cytological data from the lethal chromosomes obtained from the male and female hybrids suggest that there are at least three breakage sites in the 18F-20A region; one at the distal end, one in 19C and one at the proximal end.

P e1evze)zts 017 the V 6 X chromosome: ENCELS and PRESTON (1981b) have shown that P elements reside at the sites of frequent chromosome breakage in dysgenic hybrids. Applying this observation to our data implies that P elements reside in 14D1-3 and in the 18F-20A section of the X chromosome. Since we also observed some breakage at lA, 2B and 11A, these might also be P element sites. The question is resolved by in situ hybridization of a P element probe to V 6 X chromosomes. This hybridization shows labeling at sites lA, 11 A, 14D, 19A-B and 19C-D. Thus, there is good agreement between the breakage sites and the labeling sites, with the exceptions of 2B and the proximal part of the 18F-20A section. These exceptional sites do not seem to be P element loci on the V 6 X chromosome and may represent new P element sites generated during dysgenesis. Another possibility is that they contain very small elements that are not detected by in situ hybridization.

ENCELS and PRESTON (1981b) mapped the positions of the P elements on the X chromosome of the P strain known as HZ by collecting rearrangements generated in dysgenic hybrid males. These rearrangements had a common breakpoint in the bands 17C2-3, and all caused a mutation in the heldup (hdp) wings locus. Every hdp mutation that ENCELS and PRESTON studied was associated with a break in 17C2-3; the P element located at this point has recently been cloned and sequenced (O’HARE and RUBIN 1983). In contrast to the finding of ENCELS and PRESTON that every hdp mutation was associated with a break, we found that none of the 33 lethal mutations that came from dysgenic males and that affected the gene in band 19C4 was associated with a cytologically detectable rearrangement. Nonetheless, breaks in the 19C section of the V 6 X chromosome did occur in dysgenic hybrids, but they did not cause a lethal effect. One possible explanation is that the mutations in 19C4 result from the excision of a P element that naturally resides there, whereas the rearrangements result from coincident breakage at the 19C4 element and at other elements in the chromosome.


It is interesting to compare this situation with that of the 17C2-3 P element in the u2 X chromosome. W. ENGELS and C. PRESTON (personal communication) have observed that Beadex mutations induced by dysgenesis on the 7r2 X are associated with the excision of the 17C2-3 P element. As was mentioned earlier, heldup mutations are associated with rearrangements mediated by this element. The situation is similar to that of the 19C4 element on the v6 X chromosome, except that rearrangements involving the 19C4 element produce no obvious phenotypic effect, even opposite a deletion on the homologous chromosome.

The nature of Q strains: v6 is classified as a Q strain in the P-M system of hybrid dysgenesis. It carries P elements on its chromosomes and also possesses the P cytotype. The latter is a condition that suppresses the action of P elements. However, a complementary condition called the M cytotype exists in many strains and is able to release P elements from their inactivity. Thus, when the P elements of v6 are placed in the M cytotype by an appropriate cross, they may cause mutations and chromosome breaks. These events occur primarily at sites where P elements naturally reside on the v6 X chromosome. The P elements of v6 also destabilize snw, an allele of the singed bristle locus which is due to the insertion of a P element into the singed locus but which is incapable of self-activation (ENGELS 1979b; ENGELS 198 1 ; W. ENGELS, unpublished results; M. SIMMONS, unpublished results). All of these facts suggest that in the M cytotype some of the P elements of the v6 strain produce a transposase which then activates these elements. However, the amount of transposase is probably small compared with that produced by the P elements of the strong P strain known as 7r2. The reason is that all of the manifestations of dysgenesis are much more pronounced when the P elements of the 7r2 strain are placed in the M cytotype. For instance, the snw allele is destablized to a much greater extent (W. ENGELS, unpublished results; M. SIMMONS, unpublished results), and P element-induced mutations occur more often and at many sites, some of which were previously devoid of P elements (SIMMONS et al. 1980; SIMMONS et al. 1984). These differences might be due to a difference in the number of P elements in the two strains. However, Southern gel analysis indicates that both strains possess 30-50 P elements (BINGHAM, KIDWELL and RUBIN 1982); therefore, a simple numerical difference is probably not the explanation. Another possibility is that a small fraction of the v6 P elements might be capable of making the transposase. Restriction mapping of P elements cloned from the 7r2 genome suggests that about one-third of them are struc- turally complete and could specify the transposase (O’HARE and RUBIN 1983). A similar estimate is not yet available for the v6 elements, but two apparently complete elements have been isolated from the v6 genome (K. O’HARE, M. SIMMONS and G. RUBIN, unpublished results).

The postulated dearth of transposase-encoding elements in the v6 genome could explain why the progeny of crosses between M females and v6 males do not exhibit gonadal dysgenesis. This phenomenon is seen in the progeny of crosses between M females and 7r2 males and results from the failure of the germ line to develop. Quite probably, the transposase made by the 7r2 elements


interferes with the development of the germ cells, perhaps by inhibiting some vital aspect of their metabolism or by causing numerous lethal breaks in their DNA (ENGELS 1983). If the level of transposase were reduced, some of the germ cells might survive and be able to form the adult gonad. We conjecture that this is the case with V g and other Q strains. These strains possess many P elements but probably have only a few that are able to make the P transposase. Thus, it is likely that the concentration of transposase is sufficiently low in M x Vg hybrids that some germ cells always survive to form the adult gonad. This conjecture is an elaboration of the proposal put forth by BINGHAM, KIDWELL and RUBIN (1982) and by KIDWELL (1983) that the P elements of Q strains are defective in the potential for inducing gonadal sterility.

W. ENGELS and C. PRESTON (unpublished results cited in ENCELS 1983) have studied the frequency of gonadal dysgenesis and the level of snw instability in dysgenic hybrids made by crossing several P and Q strains with standard M strains. The two phenomena are highly correlated. In fact, a plot of snw instability us. frequency of gonadal dysgenesis is linear. Since snw instability almost certainly measures transposase activity, it appears that the frequency of gonadal dysgenesis does this also. Thus Q strains, which cause little snw instability and little gonadal dysgenesis, seem to represent one extreme in a continuum of transposase production, whereas strains such as 7r2 seem to represent the other. Accordingly, it would be proper to regard Q strains as very weak P strains rather than as qualitatively different entities.

This work was supported by grants from the National Institute of Environmental Health Sci- ences (RO 1-ESOI 960), the National Science Foundation (PCM-7903266) and the University of Minnesota Computing Center. Part of this work was completed while M. J. S. held a Bush Foun- dation Sabbatical Fellowship from the University of Minnesota. We thank JEFFREY S. COOPER, NANCY M. Cox, RHONDA F. DOLL, ERIC A. DRIER, ELIZABETH T. HAWKINS, NEAL K. KAPPLINGER and DOUGLAS R. KELLOGG for technical help; GEORGE LEFEVRE and DAVID ISH-HOROWICZ for Drosophila stocks; and WILLIAM R. ENGELS, MARGARET G. KIDWELL and JAMES F. CROW for comments on the manuscript. We also thank W. R. ENGELS for providing the materials and facilities for the i n si/u hybridizations and JOHNG K. LIM for analyzing the male-derived lethal chromosomes cytologically.

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Corresponding editor: D. L. HARTL

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