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A RECIPROCAL TRANSLOCATION IN COCHLIOMYIA HOMINIVORAX (DIPTERA: CALLIPHORIDAE). GENETIC AND CYTOLOGICAL EVIDENCE FOR PREFERENTIAL SEGREGATION IN MALES LEO E. LAGHANCE,I JOHN G. RIEMANN, AND D. E. HOPKINS Entomology Research Division, Agricultural Research Service, United States Department of Agriculture, Mission, Texas Received February 3, 1964 HE mutant strain of the screw-worm fly (Cochliomyia hominivorax) known as Brc (black R-cell) was discovered in our laboratory almost two years ago, and has been maintained for over 20 generations. Earlier studies indicated that a dominant autosomal factor was involved. Appropriate tests indicated that penetrance of the mutation was excellent, but continued selection did not result in the establishment of a pure-breeding strain. Preliminary studies of the off- spring from Brc x Brc indicated that the homozygous condition might render them viable but sterile. Further studies proved this hypothesis to be false and showed that the mutant phenotype was associated with a reciprocal translocation, that the homozygous condition was lethal, and that the aberrant segregation ratios were due to the occurrence of nonrandom segregation of the chromosomes at first meiotic metaphase in translocation heterozygotes. The genetic and cyto- logical studies leading to these conclusions are herein presented. MATERIALS AND METHODS Cochliomyia hominivorax (=Callitroga hominivorax) is an obligate parasite of warm-blooded animals. The biology and laboratory rearing procedures were discussed in several publications (MELVIN and BUSHLAND 1941; BUSHLAND 1960a, b). All flies used in these studies were reared on artificial media, allowed to pupate in sand or sawdust, and the adults were kept in wire mesh cages with honey and water as food. Colonies were maintained at 80°F. Other experimental procedures are described in connection with the tests in which they were employed. The term “Brc flies” denotes adults which have the mutant phenotype due to a dominant factor, but use of the term implies no distinction between heterozygotes and possible homozygotes. RESULTS Origin and phenotype of the Brc mutation: The mutation is characterized by having the entire R-cell in the wing completely blackened by dense pigmentation (Figure 1). The mutant flies have excellent vigor and appear normal in all other respects. The mutant strain was derived from a single male found among the progeny of irradiated flies. The mutant male was crossed to wild-type females Present address: Metabolism and Radiation Research Laboratory, Entomology Research Division, U.S.D.A., State University Station, Fargo, North Dakota. Genetics 49: 959-973 June 19G4.

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A RECIPROCAL TRANSLOCATION IN COCHLIOMYIA HOMINIVORAX (DIPTERA: CALLIPHORIDAE). GENETIC AND

CYTOLOGICAL EVIDENCE FOR PREFERENTIAL SEGREGATION IN MALES

LEO E. LAGHANCE,I JOHN G. RIEMANN, AND D. E. HOPKINS

Entomology Research Division, Agricultural Research Service, United States Department of Agriculture, Mission, Texas

Received February 3, 1964

HE mutant strain of the screw-worm fly (Cochliomyia hominivorax) known as Brc (black R-cell) was discovered in our laboratory almost two years

ago, and has been maintained for over 20 generations. Earlier studies indicated that a dominant autosomal factor was involved. Appropriate tests indicated that penetrance of the mutation was excellent, but continued selection did not result in the establishment of a pure-breeding strain. Preliminary studies of the off- spring from Brc x Brc indicated that the homozygous condition might render them viable but sterile. Further studies proved this hypothesis to be false and showed that the mutant phenotype was associated with a reciprocal translocation, that the homozygous condition was lethal, and that the aberrant segregation ratios were due to the occurrence of nonrandom segregation of the chromosomes at first meiotic metaphase in translocation heterozygotes. The genetic and cyto- logical studies leading to these conclusions are herein presented.

MATERIALS AND METHODS

Cochliomyia hominivorax (=Callitroga hominivorax) is an obligate parasite of warm-blooded animals. The biology and laboratory rearing procedures were discussed in several publications (MELVIN and BUSHLAND 1941; BUSHLAND 1960a, b). All flies used in these studies were reared on artificial media, allowed to pupate in sand or sawdust, and the adults were kept in wire mesh cages with honey and water as food. Colonies were maintained at 80°F. Other experimental procedures are described in connection with the tests in which they were employed.

The term “Brc flies” denotes adults which have the mutant phenotype due to a dominant factor, but use of the term implies no distinction between heterozygotes and possible homozygotes.

RESULTS

Origin and phenotype of the Brc mutation: The mutation is characterized by having the entire R-cell in the wing completely blackened by dense pigmentation (Figure 1). The mutant flies have excellent vigor and appear normal in all other respects. The mutant strain was derived from a single male found among the progeny of irradiated flies. The mutant male was crossed to wild-type females

Present address: Metabolism and Radiation Research Laboratory, Entomology Research Division, U.S.D.A., State University Station, Fargo, North Dakota.

Genetics 49: 959-973 June 19G4.

960 L. E. LACHANCE et al.

that produced a total of 46 offspring (30 Rrc: 16 normal). Unfortunately, all the F, males were accidentally discarded; therefore, Brc females were outcrossed to wild-type males and produced 78 Rrc:87 normal F, progeny. The normal flies were discarded and the mutant flies mated inter se. In each succeeding genera- tion the mutant flies were separated from the normal ones before crossmating could occur and the Brc flies were placed in a large stock cage for random mating. This method of selection was employed for more than 20 generations. Results are presented in Table 1.

Some of the Brc adults in the F, were outcrossed to a wild-type stock; and from the Rrc flies produced in the F, (heterozygotes) a new mutant stock was established. The results of selection in this second Rrc stock are presented on the second line beginning with the F, generation in Table 1. It is obvious from the data that about 75 percent of the flies in each generation were Rrc mutants (3 Brc:1 normal ratio). During the first 20 generations 31,439 mutant progeny were observed from a total of 42,810 flies examined (73.4 percent Rrc). This regular pattern of segregation was consistent for over 20 generations and indi- cated that failure to establish a pure-breeding strain was not due to poor or vari- able penetrance of the Brc factor.

If the Rrc phenotype were due to an autosomal dominant mutation and the homozygous condition were lethal, then mutant x mutant crosses should have produced 2 Rrc heterozygotes: 1 normal. The data in Table 1 indicate that a 3: 1 ratio was consistently obtained. This result suggested that perhaps flies homo- zygous for the Brc factor did not die, but rather were sterile, and although they survived, did not reproduce. Further tests were undertaken to test this hypothesis.

Four separate types of investigations were conducted: (a) Studies on the reproductive system of adult Rrc flies; (b) fertility studies of the Rrc flies in

I

FIGURE 1.-Normal (top) and Brc (black-R-cell) mutant wing (bottom) in Cochliomyfa hominivorax.

CYTOGENETICS O F A TRANSLOCATION

TABLE 1

96 1

Inheritance of the Brc phenotype. Proportion of Brc flies among the progeny from Brc x Brc crosses

Number of progeny

Generation Total Brc Normal Percent Brc

3 4 5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

139 360 925

1,161 166 154 95 1 345

1,300 1,460 2,414 2,425

203 2,609

964 1,572 1,160 1,205 1,690 1,550

1,078 2,029

646 1,225

500 1,793 2,185 1,932

794 1,501 1,333 1,512 1,693

1,846

117 270 682 81 1 1 22 105 709 244 974

1,089 1,773 1,784

135 2,021

71 7 1,164

886 893

1,240 1,191 1,363

747 1,458

4.53 854 377

1,292 1,560 1,365

571 1,127

975 1,130 1,240

22 90

243 350 4-4 49

242 101 326 361 641 64 1 68

588 247 408 274 312 450 359 483 331 571 193 371 123 501 625 567 223 374 358 382 463

84.2* * 75.0 73.7 69.9* 73.5 68.2 74.6 70.7 74.9 75.1 73.4 73.6 66.5* 77.5** 74.4 74.0 76.4 74.1 73.4 76.8 73.8 69.3* 71.9* 70.l* 69.7* 75.4 72.1* 71.4' 70.7* 71.9* 75.1 72.6 74.7 73.2

Total 42,810 31,439 11,371 73.44

* Significantly lower than 3 Bn : 1 Normal at 0.05 level. * * Significantly higher than 3 Brc : 1 Normal at 0.05 level.

various outcrosses; (c) studies of the progeny produced from single pair matings; and (d) cytological studies on the Brc mutants.

Studies on the reproductiue systems of Brc mutants: The reproductive systems of 109 sexually mature Brc females, selected at random, were examined. A few flies had undeveloped ovaries, but their number was not higher than expected in wild-type flies. It was concluded that Brc females had completely normal ovarian

962 L. E. LACHANCE et a1

development and that maturation of the oocytes proceeded normally and as rap- idly as in wild-type females.

In a companion test, the testes of 116 Brc males were examined and 104 were found to be well developed, while the other 12 possessed one normal and one completely undeveloped testis. In another test, the testes were removed from adult Brc males, squashed in saline, and examined with a phase contrast micro- scope. In 77 out of 79 males the testes appeared to contain a normal supply of mature motile sperm. It was concluded that the reproductive system had devel- oped normally and produced normal quantities of mature sperm.

Progeny tests in Brc x wild type outcrosses: In order to test whether all Brc adults taking part in a fertile mating were heterozygotes ( B r c / + ) , results of mating Brc males to wild-type females or the reciprocal cross were studied. In these tests male to female ratios of 2: 5, 1 : 4, and 1 : 1 were utilized (see Table 2). An examination of the progeny records in Tests 2, 4, and 5 (Table 2) showed that not a single pair mating of Brc to wild-type flies produced exclusively Brc progeny. The pooled results of numerous cultures indicated that in outcrosses involving a Brc and a wild-type fly the mutant was always heterozygous for the genetic factor, and that the mutant progeny produced averaged between 48 and 51 percent. In addition, whenever Brc females were involved in an outcross with normal males, the cultures were often lost and fewer progeny were produced. For example, in Test 4, and 61 egg masses collected to initiate cultures, only 27 eventually produced progeny, with an average of 46 adults developing per cul- ture. Test 3 produced a better yield of fertile cultures, but since the eggs of five females were pooled for each culture, many of the females could have been sterile and a fertile culture would still have been produced. The females in this test produced an average of 30 progeny. In comparison, when Brc males were out- crossed, 17 out of 21 cultures produced progeny, with an average of 72 adults developing per culture. These values clearly indicate that fertile Brc flies are heterozygous for the mutant factor and that these females are always less fertile than the males.

Fertility studies: Estimates of the fertility of Brc x wild-type matings or Brc X Brc crosses were obtained by means of egg hatchability studies. In these tests (summarized in Table 3 ) each female was allowed to oviposit an egg mass in a separate vial. After oviposition, each female was checked for the presence of sperm in the spermathecae and only egg masses from fertilized females were used for hatchability studies. In Trials 1 to 3, the egg masses from fertilized fe- males were pooled and separated in 1 percent NaOH. In each random sample, about 1,000 eggs were removed, plated on a damp cloth in five petri dishes, and counted. The hatchability was determined 24 hours later. In Trial 4, the egg masses from individual females were plated and counted separately to determine the number of eggs produced by, and the fertility of, each female. Results are presented in Table 3.

In the column headings in Table 3, Brc flies are represented as heterozygotes (Brc/+) , an assumption which seemed justified from the data in Tables 2 and 3. It is apparent from Table 3 that certain crosses involving Brc mutants were less

CYTOGENETICS O F A TRANSLOCATION

TABLE 2

Progeny tests with Brc x wild type outcrosses

963

Cross Number of cultures Number of progeny Test

number Males X Females Started Fertile Normal Brc Percent Brc ~ ~~

2 : 5 1 Brc : Normal 10 9 1,651 1,513 47.5

1 : 4 2 Brc : Normal 10 7 657 620 48.6

2 : 5 3 Normal : Brc 10 10 742 746 50.1

1 : l 4 Normal : Brc 61 27 630 607 49.1

1 : l 5 Brc : Normal 21 17 598 627 51.2

viable than others, but that for each type of cross hatchability results of all four replicates agreed very closely. Each type of cross resulted in a characteristic de- gree of fertility.

In computing an average fertility for each type of cross, the data from Trial 1 were omitted since the wild-type males used in this test were of very low vigor and fertility. Both crosses in which wild-type males were used had lower fertility than was obtained from all subsequent tests.

In crosses of Brc males to normal females, the average egg hatchability was

TABLE 3

Egg hatchability from crosses of Brc mutants

Brc Brc + + -?? x --dd - -dl3 x -?? + + + + + + + + - x - - x - Brc + Brc + Type Of cross

Number Percent Number Percent Number Percent Number Percent Trial eggs hatch eggs hatch eggs hatch eggs hatch

1 961 28.0 1,972 58.6 2,150 32.8 883 62.3 2 1,622 38.5 1,880 67.8 1,638 31.0 1,698 96.8 3 1,063 37.8 956 70.5 1,036 31.9 907 92.8

4 4,503 50.9 5,4U5 60.1 5,259 39.0 5,407 93.8 Number of

egg masses 19 24 22 21 Average number of

eggs per female 237.0 225.2 239.0 257.5 Range of hatch+ 29.2-61.5 7.0-77.8 30.1-51 .O 85.4-100

Grand average egg hatch* 49.0 66.9 38.7 . .

* Grand average hatchability based on pooled data from Trials 2, 3, and 4 corrected for the mean control hatchability

t Range in the hatchability of the individual egg masses from single pairs. for those three trials.

964 L. E. LACHANCE et a1

67 percent. Of 24 egg masses examined in Trial 4, hatchability values in 23 ranged from 41 to 78 percent, but a single egg mass had a low hatch of 7 percent. When Brc females were crossed to wild-type males the average hatchability was 49 percent; 19 egg masses were studied separately in Trial 4 and 2 had 29 and 39 percent hatch, 13 ranged from 40 to 55 percent hatch, and 4 ranged from 55 to 62 percent hatch. Crosses involving Brc x Brc flies were the least fertile. In these tests the average egg hatch was 39 percent, and among the 22 egg masses studied separately in Trial 4, 11 ranged from 30 to 39 percent hatch and 11 from 40 to 51 percent hatch. I t is clear that the Brc females are consistently less fertile than the males and that a certain level of fertility is characteristic of each type of cross. These results indicate that a fairly regular mechanism is operative in determining fertility of the Brc mutants.

The combined data from Tables 2 and 3 indicate that Brc flies are semisterile. Partial sterility could not be attributed to lethality of Brc homozygotes since in Brc x wild-type outcrosses the fertility was significantly and consistently lower than in the controls, although Brc homozygotes are not formed in such crosses.

In tests conducted to determine the linkage relationships of different mutations in the screw-worm fly (unpublished data), it was discovered that the Brc factor was linked to the autosomal recessive fused ( fu ) (21.3 percent crossovers), and to another autosomal recessive yellow eyes ( y ) ( 17 percent crossovers). On fur- ther testing for recombination frequency between fused and yellow, independent assortment was obtained. The alteration of linkage relationships between auto- somal mutations suggested that a chromosome translocation might be involved. This hypothesis was confirmed in the cytogenetic analysis of the Brc mutants.

Cytogenetic studies: Cytological investigations were conducted on the repro- ductive cells of males. Spermatogonia and primary spermatocytes were examined. Cytological studies on the females are very difficult, principally because of the extremely small size of the oocyte chromosomes. Consequently, all of our infor- mation on the cytogenetics of the Brc factors was obtained from males.

The testes were dissected from pupae two to three days old and stained with aceto-orcein after brief fixation in Carnoy’s solution. Of 103 male pupae ex- amined from a Brc x Brc cross, four could not be scored and 51 had typical trans- location crosses (see Figure 2C and D) in the primary spermatocytes. Few of the spermatogonial figures exhibited definite crosses (Figure 2B), although the chro- mosomes involved in the translocation were always closely associated. The re- maining 48 pupae had normal chromosome configurations (see Figure 2E and F) . Since the number of pupae with normal chromosomes significantly exceeded the 25 percent normal adults usually found in the Brc stock, this result suggested that perhaps most Brc homozygotes survived at least to the early pupal stage. Translocation homozygotes generally exhibit normal chromosome pairing.

Emergence studies were conducted to determine whether eclosion from the pupae was less than normal, as would be expected if the Brc homozygotes died in the pupal stage. Normal emergence is usually 94 percent or higher. Four groups of 100 pupae from the 20th generation were randomly selected for these emer- gence studies. Pupae were held in the colony room and the numbers of flies

CYTOGENETICS OF A TRANSLOCATION 965

i.

e

i

F

--

e FIGURE 2.-Wiltl-typr and nrc translocation chromosome complexes in the screw-worm fly.

3200 X. A. Wild-type spermatogonial pre-metaphase. B. Spermatogonial metaphase from Brc heterozygote. C and D. Metaphase I in primary spermatocyte from a Brc heterozygote. (Note typical translocation crosses.) E. Metaphase I in primary spermatocyte from a wild-type adult fly. F. Metaphase I in primary spermatocyte from an adult BIT homozygote, a rare class.

966 L. E. LACHANCE et al

emerging scored. In these four groups of pupae, emergence ranged from 79 to 88 percent (83 percent average), and 77 percent of the adults were Brc flies. In crosses of Brc x Brc, if normal and translocation homozygotes are formed in equal numbers and all translocation homozygotes die in the pupal stage, then 75 percent emergence would be the maximum expected. However, if some of the translocation homozygotes perished prior to pupation, then emergence values higher than 75 percent could be expected.

Aceto-orcein squashes were also prepared from the testes of 84 adult Brc males, aged from one to two days. Ten of the preparations could not be scored because of their poor quality or the near-death condition of the males selected; but of the remaining 74 preparations, 70 (94.3 percent) were translocation heterozy- gotes. The other four males had a “normal” chromosome complex of six bivalents (see Figure 2F). Generally, a translocation homozygote cannot be distinguished from a normal pair of chromosomes. With these four cytologically normal Brc males, however, the translocated chromosomes could be identified with careful study since one pair of autosomes was shorter than normal and another pair of autosomes was longer. Obviously, a few Brc homozygotes survive to the adult stage, at least in the males.

Additional cytogenetic studies on spermatogonial cells (Figure 2A and B) and numerous primary spermatocytes revealed that the Brc translocation involves the long arm of chromosome I1 and the short arm of chromosome IV, with breakage points near the centromeres in each chromosome (see Figure 3). The chromo- some numbering system employed in Figure 3 is based on the idiograms and terminology of BOYES (1 961 ) . An examination of this figure shows that as a result of chromosome breaks in chromosomes I1 and IV followed by reciprocal exchange of almost entire chromosome arms, two new chromosomes were formed. The shorter of these is 8.6 percent shorter than autosome I1 in wild-type flies and the longer is 6 percent longer than autosome IV and almost indistinguishable from chromosome VI, which is the longest chromosome in the genome (see Figure 2F).

DISCUSSION

From the studies reported we conclude that the Brc mutation is associated with a reciprocal chromosome translocation involving two autosomes. The homozy- gous condition is usually lethal in the pupal stage and the few homozygous adult

escapers” do not reproduce, either because of death before sexual maturity or low vigor. Flies heterozygous for the translocation exhibit the mutant phenotype (Figure 1 ) as do the rare homozygotes. GLASS (1934) described several domi- nant visible eye-color changes associated with translocations. The fertility of both male and female heterozygotes is reduced. There remain the questions why the Brc males are more fertile than the females and why crosses of Brc x Brc regu- larly produce 75 percent mutant progeny. The consistency with which this ratio is obtained indicates that a very regular mechanism is involved in determining the fertility of the Brc flies and the type of progeny they will produce. The ex- planation can be found in the production of different kinds of gametes by the

CYTOGENETICS O F A TRANSLOCATION 967

translocation heterozygotes. We shall concern ourselves mainly with this point. During meiosis, chromosome pairing is between homologous loci; thus, the four

chromosomes in a translocation heterozygote form a complex configuration which resembles a cross (see Figure 2B, C, D, and SWANSON 1957; WHITE 1954) with the pairing partners exchanged at the point of the translocation breaks. At ana- phase I, the opening of this configuration and its position on the plane of the spindle can proceed in three possible ways which have been diagrammed and discussed in many publications (see HADORN 1961, pp. 86-88 and SWANSON 1957, pp. 174-176). The three possibilities are: (1) Alternate disjunction, in which the homologous centromeres go to opposite poles in a zig-zag fashion pro- ducing viable gametes which are either completely normal or contain translo- cated chromosomes. (2) Adjacent-1 disjunction in which homologous centro- meres go to opposite poles in a block-type separation. This type of segregation leads to the production of gametes which contain duplications for an entire chro- mosome segment and are deficient for an entire chromosome segment. (3)Adja- cent-2 disjunction in which the homologous centromeres go to the same pole. This last type of segregation also produces duplication-deficiency type gametes.

Thus, six different chromosomal combinations can be allocated to the gametes. Only two of these are normal in that they contain all chromosomal loci, and these combinations are derived from an alternate disjunction type of segregation. In the other types of segregation, certain loci are missing (deficiency) while at the same time others are duplicated. Such germ cells produce zygotes with un- balanced sets of chromosomes. Consequently, parents heterozygous for a recipro- cal translocation produce germ cells transmitting deficiencies and duplications in addition to normal gametes. Although the duplication-deficiency gametes are inviable in plants because the gametophyte is unable to survive such genic changes, they can survive in animals. Chromosome imbalance apparently does not affect the viability of eggs or sperm of Drosophila (BRIDGES 1916) or of Coch- liomyia (Figure 3) when they are in the haploid state. Certain offspring of trans- location heterozygotes become hypodiploid for some blocks of genes and hyper- diploid for others, a condition which is usually lethal. However, viable individuals arise from the union of the normal gametes or from the union of two duplication- deficiency gametes, provided they are complementary types. What is absent or duplicated in one gamete is compensated for in the other, to give a full diploid set of genes (see Figure 3).

If the three types of segregation occurred at random, a translocation would lead to inviability in approximately two thirds of the gametes (66 percent steril- ity). It is obvious that the fertility of the Brc males was considerably higher. Average fertility of Brc males was 66 percent while that of the Brc females was 50 percent (Table 3 ) . Significantly higher fertility of male translocation hetero- zygotes compared to females was also observed by PIPKIN (1940). We conclude that disjunction in the Brc translocation heterozygotes is not random and that a mechanism is operative which regulates disjunction, but that this mechanism is different in males than in females. Brc males produce fewer duplication-defi- ciency gametes than females, as can be seen from their higher fertility. The re-

968 L. E. LA CHANCE et a1

mainder of the discussion will deal with the differences in meiosis between males and females, with an attempt to assign numerical frequencies to the different types of gametes produced by each sex.

We assume that adjacent-2 chromosome segregation does not feature impor- tantly in the fertility of the Brc flies. This assumption seems justified since homologous centromeres usually go to opposite poles and our studies on spermato- genesis in normal and Brc males indicated that adjacent-2 disjunction was not very common. If chromosome disjunction is more or less at random, then one has to assume that orientation of the centromeres on the spindle is nonrandom in order to account for the fertility levels observed. Therefore, we shall consider only alternate and adjacent-1 types of disjunction as possible types of segregation. If both occurred in equal frequency, 50 percent of the gametes would be fertile. In male Brc flies hatchability studies showed that at least 66 percent of the gam- etes were fertile. Evidently, alternate disjunction was, on the average, twice as frequent as the adjacent-1 type. As a consequence of this preferential segrega- tion, the frequency of four types of gametes produced by Brc males was .33 normal: .33 translocations: .33 duplication-deficiency (see Figure 3).

Preferential recovery of certain chromosomes is not throughly understood, al- though such preferential segregations in translocation heterozygotes have long been recognized, especially among plants. (See SWANSON 1957, pp. 320-327 for more detailed discussion of preferential segregation in various organisms.) Pref- erential segregation favoring the movement of alternate chromosomes to opposite poles has also been observed in maize (BURNHAM 1934,1962). In many instances, the type of segregation is under genetic control.

There are several factors which would appear to favor alternate disjunction in Brc males. Both WHITE and SWANSON (and others) stressed that the kind of segregation will be largely influenced by factors such as the length of the translo- cated segments, chiasma frequency in the translocation cross, and the dimensions of the chromosomes relative to that of the spindle. SWANSON states: “Presumably the more flexible the ring the greater would be the opportunity for maneuvering the chromosomes to give alternate disjunction” and elsewhere: “Flexibility of the ring would improve the chances for proper orientation,, and one consequently finds that those forms having a high degree of alternate segregation possess few or no interstitial chiasmata.” Since in male screw-worm flies no chiasma formation or crossing over occurs, the complex is quite flexible. Presumably this flexibility ol the Brc translocation complex in males favors alternate segregation over ad- jacent-1 disjunction. The results suggest that with this type of translocation, the maximum frequency of alternate disjunction is twice that of adjacent-1 separation.

For Brc females 49 percent of the gametes were fertile (Table 3 ) , which was exactly that expected on the basis of random occurrence of alternate us. adja- cent-1 disjunction. In the females, however, chiasmata are formed and crossing over takes place, which somewhat restrict the flexibility of the translocation cross. Apparently, in this less flexible situation, with chiasmata formed in the arms of the translocation, alternate and adjacent-1 type segregations are equally

CYTOGENETICS O F A TRANSLOCATION 969

frequent. Equal frequency would lead to the production of gametes half of which are fertile (balanced types). This expected fertility is in excellent accord with the hatchability data in Table 3.

According to results of our analysis, the breakage points involved in the Brc translocation were very close to the centromeres, which condition would lead to a very small “interstitial segment.” If crossing over did occur in the inter- stitial segment, all gametes formed as a result of adjacent-1 disjunction would not be of the duplication-deficiency type, but half of them would be of the balanced type. However, alternate disjunction following crossing over in the interstitial segment is expected to produce gametes half of which are duplication- deficiency types (see BURNHAM 1949, 1962). Consequently, we feel that in the Brc females crossing over is mostly restricted to the chromosome arms and occurs rarely in the interstitial segment; but when crossing over does occur in the inter- stitial segment, it has little bearing on the fertility of the females since the equal frequency of adjacent-1 and alternate-type disjunction interact to maintain the fertility of the females at 50 percent. The feasibility of this hypothesis is sup- ported by the 49 percent hatchability observed in Brc female x wild-type male crosses.

In Drosophila, a definite correlation exists between the type of disjunction and the amount of crossing over taking place in the translocated segments (BROWN 1940). GLASS (1935) also found that the segregation of translocation heterozygotes in Drosophila was not at random, being decidely skewed in the direction of viable combinations.

Thus, we arrive at the hypothesis presented in Figure 3. The translocation complex with the normal and translocated chromosomes is presented. Each chro- mosome arm was assigned a letter to facilitate representation of the four chromo- somes as they assort in pairs in the gametes. The only restriction that this hy- pothesis imposes on random assortment is that adajacent-2 disjunction is absent or rare (or that orientation of the centromeres is nonrandom) and that the males produce twice as many viable gametes as duplication-deficiency gametes because of the greater frequency of alternate versus adjacent-1 disjunction. The females, on the other hand, produce equal numbers of viable gametes and duplication- deficiency gametes. As previously mentioned, in insects the gametes bearing duplications and deficiencies are able to function in the formation of zygotes.

Examination of the Figure 3 discloses that 10 of the 16 possible zygotic com- binations are duplication-deficiency zygotes and consequently lethal. Therefore we expect 58.4 percent of the zygotes formed to perish before hatching, and in our tests we observed 61 percent lethality before hatching. One combination produces a Brc homozygote (8 percent) which is generally lethal in the pupal stage. Since the Brc homozygotes usually survived to the pupal stage, we expected that the cytogenetic examination of the spermatocytes in the pupae would reveal approximately 40 percent cytologically normal, and 60 percent translocation heterozygotes. Our studies on 99 pupal testes revealed that 52 percent were trans- location heterozygotes (not significantly different from the expected value at 0.05 percent level).

970 L. E. LACHANCE et a1

A A C C B A C

B D I1 IV

PATERNAL GAMETES ALTEWATE 4DJACENT- I DISJUNCTION DISJUNCTION

3 3 3 AB CD I 3 3 3 AD C B I 167 AB C B I 167 AD CD I

0 8 3 3 0833 0418 0418 AD CO

AD AD AD AD CB Bre Homo

Brc Het Pupal Lethal d d d d 0 8 3 3 1 0833 1 0418 I 0418

1 AB

AB ;; 1 AD 1 AD rl 1 AB AB AB AB CB

d d d d Bre Het

AB CO AD AB CB AD AD CO AD CD AD CD AD CD

d d d d Brc Het d d

d d 0833 0 8 3 3 0418 0418

FIGURE 3.-Model for reproduction in the Brc translocation stock. Top row: diagram of normal and translocated chromosomes. Lower portion: Mating chart showing type and frequency of gametzs formed by males and females and the frequency of resulting zygotic combinations with the expected viability of each combination . (d.d. = duplication-deficiency zygote, lethal before hatching; Brc Homo. = translocation homozygote, pupal lethal; Brc Het. = translocation heterozygote, viable and partly fertile).

The five other zygotic combinations shown in Figure 3 are viable and would produce either normal flies or Brc translocation heterozygotes. To sum up the expected values based on this hypothesis: Among the zygotic combinations sur- viving to the adult stage we expect 75 percent to produce Brc flies. This value is in excellent agreement with the observed results given in Table 1.

Consequently, we favor the hypothesis presented in Figure 3 as the simplest one which fits all of the observed data. There is always the possibility that more complex factors, such as those found in studies on Drosophila, may actually be operating. For example, abnormal segregation ratios in Drosophila males carry- ing the Bar-Stone translocation were shown to be influenced by modifiers located on the Y chromosomes and on the autosomes, as well as by temperature (ZIM- MERING 1960; ZIMMERING and PERLMAN 1962). Preferential segregation favor- ing the recovery of certain chromosomes is an example of chromosomal meiotic drive ( SANDLER and NOVITSKI 195 7).

SUMMARY

A dominant mutant factor (Brc, black-R-cell) in the screw-worm fly, which affects the pigmentation of a wing cell, is associated with a reciprocal transloca- tion involving autosomes I1 and IV. The mutant strain was studied for over 20

CYTOGENETICS O F A TRANSLOCATION 971

generations. In crosses of Brc x Brc the ratio of mutant to normal progeny is always 3: 1.

Translocation heterozygotes are viable and partially fertile, but translocation homozygotes usually survive to the pupal stage and die before emergence. A few adult male homozygotes were found; these had the mutant phenotype and exhibited normal chromosome pairing in the spermatocytes. They were of low vigor or completely sterile.

Heterozygous males are more fertile than females. Analysis of the fertility of Brc flies in all possible crosses suggests that in the males preferential segregation of the chromosomes at first meiotic division favors alternate disjunction with a consequent increase in the fertility of the males over the expected value. Hetero- zygous Brc females produced gametes, half of which were viable.

It is suggested that partial sterility of the heterozygotes, lethality of the homo- zygotes, and preferential segregation in the males are factors in this stock of mutant flies which interact to consistently produce about 75 percent mutant off spring.

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