Dec 26, 1985 - Genetics. Nonrandom segregation of centromeres following mitotic recombination in Drosophila melanogaster. (chromosome behavior/mitosis).
Proc. Nadl. Acad. Sci. USA Vol. 83, pp. 3900-3903, June 1986 Genetics
Nonrandom segregation of centromeres following mitotic recombination in Drosophila melanogaster (chromosome behavior/mitosis)
SERGIO PIMPINELLI*4 AND PEDRO RIPOLLt *Dipartimento di Genetica e Biologia Molecolare, Universiti di Roma, 00185 Rome, Italy; and tCentro de Biologia Molecular CSIC-UAM, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spain
Communicated by Dan L. Lindsley, December 26, 1985
mitotic recombination, a single answer: recombinant chromatids nonrandomly migrate to opposite poles, indicating that x-segregation is favored over z-segregation after mitotic recombination.
Mitotic recombination is widely used in ABSTRACT Drosophila as a technique to study genetic and developmental problems. It has been generally assumed that, following mitotic exchange between homologous chromatids during the G2 stage, the centromeres attached to the chromatids involved in the exchange segregate randomly. As a result, two equally frequent types of segregation, yielding genetically different products, are produced. However, when epidermal or enzymatic cellmarker mutants are used, only one type of segregation gives rise to marked cells. In the present work we test this assumption of random segregation using cytological markers. With cytological markers, larval neuroblast cells resulting from mitotic recombination exhibit recognizably all possible products from mitotic recombination. We find that one type of segregation is favored, in that, after mitotic recombination, the centromeres attached to the chromatids involved in the mitotic exchange preferentially migrate to opposite poles during anaphase. This favored segregation could be the result of exchange between previously oriented chromatids or could be due to the effect of the exchange upon subsequent orientation of homologous chromosomes. In either case, frequencies of mitotic recombination have been overestimated in the past.
MATERIALS AND METHODS As a cytologically marked chromosome, a derivative of the
XDYP element of T(X; Y)N29 (4) was used. To this element, Kennison attached by recombination the distal end of YS (for mutations and chromosomes, see ref. 5). The marked X chromosome is formed by the distal end of YS, the euchromatic region of the X chromosome in normal sequence, most of the proximal heterochromatin of the X chromosome including its nucleolar organizer, and the proximal part of YL, the centromere of the Y chromosome, and the proximal part of YS with its nucleolar organizer. For a detailed description of the Y chromosome, see refs. 4 and 6. The fluorescent properties of the different heterochromatic regions used as markers can be found in refs. 4 and 6-8. Two properties make these heterochromatic blocks useful cell markers: (i) using appropriate staining techniques, they fluoresce brightly, whereas euchromatic regions remain dull, (ii) at metaphase, sister heterochromatic blocks always appear tightly apposed, whereas euchromatic regions are separated. As a consequence, chromosomes with terminal heterochromatic blocks cytologically resemble ring chromosomes. In particular, in a Hoechst 33258-stained metaphase of a female heterozygous for a marked and normal X chromosome (see Fig. 2a), the marked X chromosome has a small fluorescent block (SFB) attached to the distal end of the long arm and, proximally, a large brightly fluorescent block (LFB), which is formed by the normal proximal heterochromatin of the X chromosome and parts of YL and YS. The normal X chromosome shows a brightly fluorescent proximal block of intermediate size (IFB). Females homozygous for a cytologically normal X chromosome were crossed to T(X;Y)N29, y y+ w f BS males. Cultures were kept at 24 -+- C until the first pupae appeared. Cultures were then irradiated with 1500 rads (1 rad = 0.01 gray) of x-rays (180 kV, 6 mA, 3 mm Al, and 100 rads/min). After 10-11 hr, brains were dissected from third instar larvae, cultured in colchicine for 90 min, and stained with Hoechst 33258 (7). Under these conditions, cells irradiated mostly in the S-G2 stages go through one complete cell cycle (9), permitting all chromosomes involved in exchange to segregate. They were then analyzed in the next metaphase. In addition, most of the cells with aberrations are eliminated
In many higher organisms there is a certain frequency of homologous recombination that occurs in mitotic (somatic) cells (1). There are three possible types of centromere segregation after mitotic exchange in the G2 stage, which Stern (2) called x-, y-, and z-segregation; they are shown in Fig. 1. y-Segregation is equivalent to a reductional segregation and it does not happen with appreciable frequency (1-3). It gives rise to clones homozygous for marker genes that are not distal to a mitotic exchange. Of the other two types of segregation, only one (x-segregation) gives rise to cells that can be visualized using cuticular or enzymatic markers in conventional clonal analysis. Because marked clones are found in individuals heterozygous for cell-marker mutations, there is no doubt that x-segregation takes place. Despite the difficulty of measuring type z-segregation, owing to the absence of genetic consequence, it became generally accepted that centromere segregation occurs at random: after mitotic exchange both recombinant chromatids migrate to the same pole half of the time (yielding no recognizable clones), while the other half of the time chromatids involved in the exchange move to opposite poles, producing marked clones. The implication is that the actual frequency of mitotic recombination is twice that of marked somatic clones. Chromosome segregation during mitosis is a central problem to cell division-a variety of mechanisms might be involved; we have used cytological markers to address this question. We designed an experiment that has given us, in addition to the cytological evidence of the occurrence of
Abbreviations: SFB, small fluorescent block; LFB, large fluorescent block; IFB, intermediate-sized fluorescent block. tTo whom correspondence should be addressed at: Dipartimento di Genetica e Biologia Molecolare, Universita di Roma "La Sapienza," Ple Aldo Moro, 5, 00185 Rome, Italy.
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(10). However, since there are cells with longer-than-average cycles, a fraction of metaphases scored derive from cells irradiated in the G1 stage of that same cycle.
RESULTS Fig. 1 shows diagrammatically the classes of marked cells expected after mitotic recombination during the four chro-
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matid stages of the cell cycle in the genotype we have used. The following cell types are expected: x-Segregation, A-type: characterized by one X chromosome carrying a LFB and a SFB and the other with an IFB and a SFB. This cell type is unique to this type of exchange and segregation. x-Segregation, B-type: recognized by one X chromosome with a LFB and the other with an IFB. This configuration can also arise from deletion of the SFB of the marked chromo-
FIG. 1. Different cell types expected after mitotic recombination in the G2 stage followed by three kinds of segregation of centromeres in cells heterozygous for genetic (a, b) and cytological markers (LFB, IFB, SFB). (In the first row a cell division without mitotic recombination is presented. In this case, the daughter cells have unique genetic constitution and metaphase configurations.) In conventional clonal analysis, a and b would be two recessive alleles of cuticular or enzymatic cell markers and + would be their wild-type alleles. LFB, IFB, and SFB, large, intermediate, and small fluorescent heterochromatic blocks. Solid circle = Y centromere; open circle = X centromere; thin lines = euchromatin; thick lines = heterochromatin; 1-4, centromeres; ME, mitotic exchange. Note that by cuticular markers it is possible to recognize only cells derived from x-segregation because of their homozygosity for recessive alleles. By cytological markers, on the other hand, it is possible to clearly recognize at least one of the two products of each segregation.
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some or translocation of this chromosome with any of the autosomes during the G1 and G2 phases. Only G1 translocations and, presumably in half of the cases, G2 exchanges are recognizable. y-Segregation, C-type: characterized by a chromosome with a heterochromatic fluorescent block at each side-one large (LFB) and the other small (SFB) and another chromosome with a LFB. y-Segregation, D-type: characterized by a chromosome with a heterochromatic fluorescent block at both sides-one of intermediate (IFB) and the other of small (SFB) size and a normal chromosome with a fluorescent block of intermediate size. z-Segregation, E-type: characterized by a chromosome with heterochromatic fluorescent blocks of different sizes (LFB and SFB) and a normal chromosome with a fluorescent block of intermediate size (IFB). This cell type is identical to the parental nonrecombinant cell type. z-Segregation, F-type: recognized by a chromosome with a SFB and an IFB and a chromosome with a LFB. This configuration is not unique to this type of exchange and segregation: it can also arise from an exchange between the chromosomes during the two-chromatid (Gl) stage. Results obtained after irradiating female larvae of the genetic constitution shown in Figs. 1 and 2 are presented in Table 1. Two different sets of experiments were performed. In experiment 1, we included in the B-type category also cells that were the results of G1 and G2 exchanges, whereas in experiment 2 these cells were recognized and excluded. From the difference between the two experiments, it is possible to estimate roughly the relative frequency of G1 and recognizable G2 exchanges among B-type cells, comparing them with the total number of A- and F-type cells. From the proportions of A:B:F in experiment 2 we infer that 73 B-type cells in experiment 1 are attributed to G1 and G2 exchanges between the marked chromosome and any autosome. As a confirmation, 724 metaphases were scored and this event was recorded: 32 B-type cells due to translocation were found among 62 B-type cells. First of all, from these data, we do not find any clear example of cells deriving from y-segregation. This confirms that y-segregation is practically nonexistent. We have observed all of the configurations derived from x- and zsegregations (see Fig. 2). There are two cell types (A and F) that are very important for the discussion of the data: cells were assigned to these cell types only when classification was unambiguous. Assuming that centromeres segregate at random after mitotic recombination and considering that (i) a fraction of cells scored was irradiated in the G1 phase and (ii) SFB deletions and some X-autosome exchanges are not recognized, B-type cells are expected to be more frequent than F-type cells and these more frequent than A-type cells. As expected, B-type cells are more frequent than either F- or A-type cells. However, contrary to what was expected after random segregation of centromeres, A-type cells are found about twice as frequently as F-type cells. This difference is statistically highly significant. Actually, the difference is Table 1. Number of metaphases with different cell types resulting from different chromatid segregations after mitotic recombination Cell type A B E F Exp. 1 3206 38 141 18 2 3086 27 47 12 Cell types E, A, B, and F are described in the text and in the legends to Figs. 1 and 2.
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FIG. 2. Examples of Hoechst 33258-stained metaphases showing the microscopic appearance of the different cell types diagramed in Fig. 1. (a) Metaphase configuration of a no-exchange cell heterozygous for a cytologically marked and a normal X chromosome. This configuration and the E-configuration due to z-segregation following mitotic crossing-over are the same. The arrows indicate the cytological markers. (b) Partial metaphase showing a chromatid exchange between the two X chromosomes induced by x-rays in the S-G2 phases of the cell cycle. The arrows point to the recombinant chromatids. (c and d) A-configurations due to x-segregation following mitotic crossing-over. Both X chromosomes possess the small fluorescent block (arrows). (e) b-configuration due to x-segregation. In this case both X chromosomes show free euchromatic ends. (f) F-configuration after z-segregation. The SFB is moved from the marked X chromosome to the normal one (arrow). X = X chromosomes; c = centromere; for LFB, IFB, and SFB, see text and the legend to Fig. 1.
even greater if one considers that a fraction of the F-type cells is not the result of mitotic recombination in the S-G2 stage but of translocation in the G1 stage. In fact, this cell type often appears together with other aberrations clearly due to irradiation in the G1 stage, such as dicentric chromosomes accompanied by reciprocal acentric fragments. To avoid introducing a bias while collecting data, no attempt was made to record these cells as different from the rest of F-type cells. On the other hand, the cell A-type configuration described above could be mistaken for cells showing, in addition to the marked X chromosome, the other one with a cytological appearance of a ring chromosome due to one G1 exchange between this chromosome and the small chromosome 4. An asymmetrical exchange would result in a dicentric chromosome and a symmetrical exchange in a translocation. This event can be distinguished cytologically because (i) the heterochromatin of chromosome 4 fluoresces more brightly than that of distal blocks of the marked X chromosome, (ii)
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in most cases one chromosome 4 should be missing from that particular metaphase plate, and (iii) in other cases the ring-shaped X chromosome should be accompanied by centric or acentric fragments with a dull fluorescence. Since there are instances when these characteristics are not met (highly condensed chromosomes, loss of fragments after squashing, etc.), we performed the following control experiment: brains of male larvae carrying a normal X chromosome (sibs of the experimental females and therefore treated under the same conditions) were checked for the presence of ring-shaped chromosomes deriving from exchanges between the X chromosome and chromosome 4. No mitotic figure of this type was found among 1000 metaphases scored, although other aberrations were recorded. Furthermore, among >6575 metaphases studied in the experimental series, only two clear cases of translocation in the G1 stage between the marked X chromosome and chromosome 4 were detected. Therefore, the event producing cells that could be mistaken for A-type cells is not frequent enough to interfere with the results. In conclusion, the results presented in Table 1 show that, at least in the genotype studied, the segregation of centromeres after mitotic exchange is nonrandom: centromeres attached to recombinant chromatids preferentially migrate to opposite poles. In other words, x-segregation is favored over z-segregation by at least a factor of 2.
DISCUSSION There are few instances in which z-segregation has been claimed to occur (see ref. 1 for review). In all of these cases, however, the results do not prove the occurrence of zsegregation and they can be explained without invoking this type of segregation. In general, if z-segregation ever happens, we lack strong experimental evidence supporting its existence. We have found cells (F-type) that could be the result of mitotic recombination during the S-G2 stages followed by z-segregation (Fig. 1). However, a fraction of these cells was the result of an exchange in the G1 stage since they also showed chromosomal aberrations unique to irradiation during this stage, such as dicentric chromosomes. We decided not to record these events as different to avoid introducing a bias in the collection of data and because it would only give an underestimate of the number of F-type cells due to G1 events without solving the problem of whether or not G2 events overyield F-type cells. On the basis of our results, we are inclined to think that after mitotic recombination, almost only x-segregation takes place. The excess of cells derived from x-segregation could be explained in two different ways. Either the exchange preferentially occurs between chromatids attached to centromeres already committed to migrate to opposite poles, or, alternatively, it is the presence of a random exchange that forces the chromosomes to orient in such a way that centromeres driving recombinant chromatids are bound to move to opposite poles. Regarding the first possibility, it has to be noted that chromatid interchanges can be induced by x-rays during S and G2 phases (9). Thus, the events leading to mitotic recombination most probably take place long before the centromeres attach to the spindle fibers. However, during prophase, homologous chromosomes appear tightly paired and this is thought to reflect intimate somatic pairing during interphase. It could well happen that the exchange takes place between those homologous chromatids that are closer together and that their centromeres are already preoriented
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so that during the subsequent metaphase they attach to spindle fibers coming from opposite poles. Alternatively, mechanical interference with chromosome orientation during prophase and early metaphase could explain the excess of A-type cells. For example, as observed, a somatic chiasma is formed after exchange and it is followed by chromosome condensation. During metaphase, the forces involved in somatic pairing of homologous chromosomes disappear while those between sister chromatids persist (see
Fig. 2b). The combined effect of both phenomena, condensation and sister chromatid pairing, could lead the somatic bivalent to adopt a spatial configuration favoring the attachment of centromeres to the spindle fibers in such a way that x-segregation is ensured. This sequence of events, however, would not apply to terminal exchange because of the absence of distal sister-chromatid pairing. Thus, there would exist the possibility of some fraction of z-type segregation following very distal exchanges. There is one important caveat to be noted. The presence of the distal heterochromatic blocks used as cytological cell markers, which stay tightly synapsed till the end of metaphase, could also play a role in the establishment of xsegregation. If these heterochromatic blocks were the only cause of the excess of x-segregation, our conclusions would only be valid for genetic constitutions similar to the one we have used. Since at the time centromeres attach to the spindle, sister chromatids are synapsed along their entire length and not only in the heterochromatic regions, we believe that our results can be generalized. Indeed, the generality of this inequality is suggested from recent data on mitotic recombination in yeast. Roman found, in fact, that also in this organism the two chromatids that are involved in the exchange usually go to opposite poles-that is, xsegregation exceeds z-segregation by a wide margin (H. Roman, personal communication). In summary, it seems exceedingly likely that following an exchange in the S-G2 stages of mitosis, segregation is not random, in that crossover chromatids segregate from one another, but sister centromeres separate as they normally do in mitosis. Although we have discussed two possibilities to explain this inequality, its basis is yet to be elucidated. Finally, whatever the cause, this implies that doubling the incidence of homozygous clones overestimates the incidence of mitotic exchange. We thank M. Gatti, D. Lindsley, H. Roman, and L. Sandler for critical reading of the manuscript. Moreover, we thank H. Roman for the citation of his unpublished results. S.P. was supported by Euratom Grant B10-E-450, and P.R. was supported by a grant from C.A.I.C.Y.T. and FIS and, in part, by a Short Term European Molecular Biology Organization Fellowship. 1. Becker, H. J. (1976) in The Genetics and Biology of Drosophila, eds. Ashburner, M. & Novitskl, E. (Academic, London), Vol. 1C, pp. 1020-1087. 2. Stem, C. (1936) Genetics 21, 625-730. 3. Baker, B. S., Carpenter, A. T. C. & Ripoll, P. (1978) Genetics 90, 531-578. 4. Kennison, J. A. (1981) Genetics 98, 529-548. 5. Lindsley, D. L. & Grell, E. H. (1968) Genetic Variations of DrQsophila Melanogaster (Carnegie Inst., Washington, D.C.). 6. Gatti, M. & Pimpinelli, S. (1983) Chromosoma 88, 349-373. 7. Gatti, M., Pimpinelli, S. & Santini, G. (1976) Chromosoma 57, 351-375. 8. Pimpinelli, S., Santini, G. & Gatti, M. (1976) Chromosoma 57, 377-386. 9. Pimpinelli, S., Pignone, D., Gatti, M. & Olivieri, G. (1976) Mutat. Res. 35, 101-110. 10. WoUT, S. (1972) Front. Radiat. Ther. Oncol. 6, 459-469.
