Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2382-2386, April 1987 Incomplete Y promote magnification in male and female Drosophila (ribosomal /ribosomal amplification) DONALD J. KOMMA AND SHARYN A. ENDOW* Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710 Communicated by D. Bernard Amos, December 12, 1986

ABSTRACT We have recently shown that magnification, on the Y that might encode a gene whose product initiates an increase in the number of ribosomal RNA genes (rDNA) in breaks in the rDNA or results in unequal pairing. Alterna- gametes produced by rDNA-deficient flies, can occur in female tively, the involvement of a limited region of the Y chromo- Drosophila if they have a Y . We now have tested some might imply a chromosomal pairing function analogous several X-Y translocation and recombinant chromosomes to to that of the collochores described by Cooper (8). determine which parts of the are necessary for The Ybb- chromosome, discovered by Schultz in 1933 (9), magnification to occur in females. Our data indicate that the has been described as being particularly effective in promot- required region is the distal part of the long arm of the Y ing magnification (1, 10, 11). In addition to promoting a high chromosome, yL. We have also used X-Y translocation chro- frequency of magnification in males carrying an Xbb chro- mosomes to study magnification of rDNA-deficient X chromo- mosome (1), it brings about the production of new bb alleles somes in males. Our data show that the region of the Y in males carrying an Xbb+ chromosome (2, 11, 12), and its chromosome from the distal end of the nucleolus organizer derivatives can bring about magnification of an Xbb chro- through the is not required for magnfication in mosome in males of a bb+ phenotype (13), suggesting that it males. The frequency of magnification in males with rDNA- is in some sense "constitutive" for magnification. No other deficient Y fragments is comparable to that produced by Ybb-, Ybb- chromosomes have been discovered. However, among a chromosome that has often been used to produce magnifica- the reciprocal X-Y translocations produced by Kennison tion in males. These results demonstrate that the Ybb- (14), there are two Y fragments that together contain all ofthe chromosome is not uniquely effective in causing magnification Y fertility factors but have no additive effect in males with an to occur in males. The results of these studies imply that Xbb chromosome, indicating that they are completely or sequences present on yL are required for magnification to almost completely lacking in rDNA. By putting both ofthese occur in females; these sequences are probably also required fragments into males with an Xbb chromosome, we can for magnification in males. Since unequal sister produce the equivalent of a new Ybb- chromosome. This exchange has been implicated as the major mechanism of procedure can be used to learn whether or not any unique ribosomal gene increase during magnification, the yL se- feature of Ybb- is required for magnification. The procedure quences required for magnification may be involved in encod- also can provide at least some information as to which parts ing or regulating products needed for sister chromatid recom- of the Y chromosome are needed for magnification in males. bination in germ-line cells. Inasmuch as both arms ofthe Y chromosome contain factors necessary for male fertility, it would be very difficult to learn Magnification is an increase in the amount of rRNA genes anything about magnification in males completely lacking a Y (rDNA) in gametes produced by severely rDNA-deficient or chromosome, but this method allows us to examine males bobbed (bb) Drosophila melanogaster, resulting in a marked lacking a significant part of the Y. reversion toward wild type in the offspring (1). The major mechanism by which this increase occurs is most probably MATERIALS AND METHODS unequal sister chromatid exchange (2, 3), but other mecha- nisms are also possible (4). We have reported (5) that Drosophila Stocks. Descriptions of all mutants used in this magnification, hitherto known only to occur in males, can work can be found in Lindsley and Grell (9). The b19 occur in severely bobbed females if the females have a Y chromosome was obtained from R. S. Hawley in March, chromosome. The frequency of magnified gametes produced 1984, and cloned in June, 1984. The y bb2 chromosome was by such females is about 1-2%. recovered after recombination in a b19/y car bb+ female. A single y bb recombinant chromosome was cloned in Novem- X-Y translocation and recombinant chromosomes can be ber, 1985. The uco3 bb chromosome was found by K. C. used to learn which parts of the Y chromosome are required Atwood (15). The y mutation was placed on the chromosome for magnification to occur in females. Such tests cannot by D.J.K. in 1974 by recombination in a bb/y car bb+ female. easily be performed in males because both arms of the Y The stock was cloned in October, 1984. chromosome are required for male fertility. Therefore, the The In(l)sc4LscSR, sc4sc8 chromosome is an inverted X fact that the Y chromosome is needed for magnification in chromosome with the nucleolus organizer deleted. It is females provides an opportunity to learn whether all of the Y referred to as X-NO in this report. The C(1)DX chromosome is required or only a small part ofit. Requirement for an entire is a compound double- with both nucleolus Y chromosome, iffound, would imply an effect similarto that organizers deleted and is denoted A-NO. Both of these of suppression-of-position effect variegation by the Y chro- variant chromosomes are described in Lindsley and Grell (9). mosome (6, 7). The requirement for a limited region of the Y The XYbb- chromosome was made by D.J.K. and is chromosome would suggest the involvement of a single described in Komma and Endow (5). It consists of an X chromosome deficient for bb but normal in sequence and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: rDNA, 18S and 28S rRNA genes. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2382 Downloaded by guest on September 27, 2021 Genetics: Komma and Endow Proc. Natl. Acad. Sci. USA 84 (1987) 2383

kl-5 kl-3 kl-2 kl-l NIt, ks-l ks-2 -v+

w19

---o vi y+

82

w28 BS

v31 BS

P7 Bs

v24 Bs

FIG. 1. Schematic diagram of the Y chromosome fragments used in this work. The top line shows a BsYy+ chromosome; the w19, w28, v31, P7, and v24 X-Y translocation chromosomes were derived from this Y chromosome. The approximate positions of the male fertility factors on the long and short arms of the Y chromosome are indicated. The X-Y translocation chromosomes are shown with solid lines indicating Y chromosome sequences and dashed lines representing regions from the X chromosome. The open and filled circles represent from the X and Y chromosomes, respectively. It is not certain whether the centromere and bb locus in scvl.Ys are from the X or Y chromosome. All Y fragments shown are either bb0 or bb- except for the scvl.Ys chromosome, of which a severely bobbed variant was used.

attached to the long arm of Ybb-. It contains all of the Y contains kl-S, is marked with Bs, and has no additive effect chromosome male fertility factors. when combined with an Xbb chromosome. X-Y Translocation Chromosomes. The translocation chro- Two other Y-fragment chromosomes were obtained from mosomes furnished to us by D. L. Lindsley were made by the Bowling Green stock center in November, 1985 (Fig. 1). Kennison (14) and further characterized and described by The scvl.Ys chromosome (18) is the result of a recombination Hardy and co-workers (16, 17). They are maintained as between the Y chromosome and the centromeric heterochro, reciprocal X-Y translocations in balanced stocks. The Y- matin of the scvl chromosome. It contains all of the Ys fragment translocations are diagrammed in Fig. 1 and are fertility factors, but it is not certain whether the centromere briefly described as follows. and bobbed locus are from the X or Y chromosome. After the T(J;Y)w19. The short arm of the Y chromosome, Ys, is stock was obtained, males were mated to C(J)DX y w brokenjust distal to the nucleolus organizer and the tip of Ys, females, and a severely bobbed LNO/Ys female carrying marked with y', is attached to the X centromere. The Ys tip the scvl.Y chromosome was selected. This chromosome was contains all of the Ys fertility factors and has no additive cloned in January, 1986, and is called scvl.Ysbb in this report. effect when combined with an Xbb chromosome, suggesting The YL y+B2 chromosome (19) is the result of a recombina- that it is completely or almost completely deficient for rDNA. tion between Ys and the distal of In(1)sc8L, T(J;Y)w28. yL is broken just distal to the centromere and ENR. It contains all of yL and the Y centromere, but not the attached to the X centromere. The yL fragment contains all nucleolus organizer. It is marked with y'. of the yL fertility factors, is marked with Bs, and has no Magnifying Crosses. All flies were grown on a standard additive effect when combined with an Xbb chromosome. cornmeal/yeast/agar Drosophila medium. Test crosses were T(J;Y)v31. yL is broken distal to kl-J, and the remainder of grown in an incubator at 250C. Magnifying flies were recog- yL is attached to the X centromere. The yL fragment nized by a severely bobbed phenotype plus the appearance of contains kl-2, k1-3, and kl-5, is marked with Bs, and has no the Y-fragment marker. Magnifying females with the additive effect when combined with an Xbb chromosome. YL.y+B2, w19Ysy+, or scvl.Ysbb chromosomes were se- T(J;Y)P7. This translocation is virtually identical to v31 verely bobbed but had wild-type (y') body color. Magnifying except that the yL breakpoint is closer to kl-2. The yL females with the w28, v31, P7, or v24 yL fragments were y fragment also contains kl-2, kl-3, and k1-5, is attached to the Bs bb. The magnifying females were mated en masse to males X centromere and marked with Bs, and has no additive effect carrying a bb0 tester chromosome, either X-NO or X'Ybb-, when combined with an Xbb chromosome. and a marked Y chromosome. Progeny were classified as bb, T(J Y)v24. yL is broken distal to the kl-3 fertility complex bbm, or bbm+ by comparing the length of the posterior and the yL tip is attached to the X centromere. The Y tip scutellar bristles and by noting the presence or absence of

Table 1. Magnification in females with Y5 fragments Score for bobbed phenotype Cross bb Y Y bb- i ii bb cT bbmf& Id % Xbb/X-No/w19 Ys YY x X-No/Y d d 715 0 1282 <0.14 XX XNo/s5cYlbb i i x tVbb-/Y d d 1611 2369 0 .0.042 gtbb-jscVl.Ysbb dd x )-No/Y Y Y 102 112 253 52 Magnifying females or males of the indicated genotype were mated en masse to tester males or females carrying a bb0 X chromosome- XNrio, Abb-, or )X-NO. Female or male progeny were scored for their bobbed phenotype on the basis of posterior scutellar bristle length. In each cross the genotype of the magnifying parent is indicated first. Totals for cross 1 do not include nondisjunctional Xbb/IXNo! Y females or Xbb/O and Xbb/w19 Ys males; totals for crosses 2 and 3 do not include nondisjunctional offspring carrying the bb+ Y chromosome. Downloaded by guest on September 27, 2021 2384 Genetics: Komma and Endow Proc. Natl. Acad. Sci. USA 84 (1987)

Table 2. Magnification in females with yL fragments Score for bobbed phenotype Cross yL bb Y99 bbm 99 CT C % Functionally complete yL (all yL fertility factors) Xbb/X-No/YL 9 9 X X-NO/Y dd B2 739 9 1366 1.2 w28 895 9 1880 1.0 Incomplete yL fragments Xbb/X-No/YL 99 x XXNo/Y dd v31 613 11 1385 1.8 P7 432 3 1348 0.7 v24 331 8 795 2.4 Magnifying females were mated to tester males carrying an X-NO chromosome and a marked Y chromosome. Female progeny were classed as bb or bbm on the basis ofposterior scutellar bristle length. Totals shown for each cross do not include nondisjunctional Xbb/X-No/ Y females or Xbb/O and Xbb/ YL males.

abdominal etching. An eyepiece micrometer was used to (Table 1). Therefore, the experiment provided evidence that measure bristle length. the w19 yS fragment is not sufficient for magnification to Magnifying males were y bb when Ybb- was used, and Bs occur in females. bb when the w19/w28 Y fragments were used. In both cases, The scvl.Ysbb chromosome, which based on its origin the magnifying males were mated to females heterozygous for should contain Ys sequences only (ref. 18; Fig. 1), showed no a B bb+ X chromosome and an X-NO chromosome. The F1 magnification in combination with the C(1)DX chromosome, females carrying the X-NO chromosome and the bbm X which is bb° and denoted A-NO, even though these females chromosome with or without a Y fragment were y, y Bs, or had a severely bobbed phenotype (Table 1). The potentially Y+ magnified sc 1iYsbb chromosomes were recovered over the XYbb- chromosome. Table 1 also shows that the scvl. RESULTS Ysbb chromosome magnifies readily in males with the X)Ybb- chromosome, producing a high frequency of bbm (52%). This Fig. 1 shows the Y chromosome fragments that were used in indicates that scvl.Ysbb is not in any sense magnification this work. For all the fragments that have no additive effect defective. with respect to the bb phenotype and are therefore either bb° These experiments demonstrate that Ys sequences do not or bb-, the general strategy consisted of making X/X/Y result in reversion of Xbb or Ybb in females. females with a y bb X chromosome, a y bb° X chromosome Magnification Occurs in Females with Functionally Com- lacking a nucleolus organizer (XNO) and the Y fragment. plete yL Chromosomes. Two Y chromosome fragments that These females were then mated to males with an XNO and contain all of yL (B2) or most of yL, including all of the yL an appropriately marked Y chromosome. Parental (magnify- fertility factors (w28), were tested for their effect on reversion ing) females carrying y+Y chromosome fragments were of Xbb in Xbb/XNO/ yL females. Both the B2 fragment, mated to males carrying the dominant Bs marker on their Y which contains all of yL and the w28 fragment, which chromosome (BWY). F1 female offspring carrying a paternal contains the yL fertility factors, produced magnification in Y chromosome were Bs and could be excluded. Parental severely bobbed females (Table 2). The frequency of mag- (magnifying) females carrying BsY chromosome fragments nification (1%) was comparable to that produced in females were mated to males with a y+Y chromosome. F1 female with a complete Ybb- chromosome (5). offspring carrying a paternal Y chromosome in these crosses Magnification Occurs in Females with Partial yL Chromo- were y+. The crosses were made in this way in order to somes. Since complete or nearly complete yL chromosome provide a distinction between female offspring carrying a fragments resulted in magnification of an Xbb chromosome, magnified Xbb chromosome with or without the Y chromo- we tested the effect ofpartial yL fragments on Xbb reversion. some fragment and nondisjunctional female offspring, which The fragments available to us, v31, P7, and v24, have would have received both X chromosomes from their mother breakpoints progressively nearer to the distal end of yL (Fig. and a Ybb+ chromosome from their father. 1). All three fragments produced frequencies ofmagnification Magnification Does Not Occur in Females with Ys Sequences comparable to that produced by a complete yL (Table 2). The Only. The Y chromosome fragment designated w19 consists differences among them, particularly the relatively low fre- ofthe tip of Ys containing all ofthe Ys fertility factors but not quency of magnification observed with P7, are probably not the nucleolus organizer (Fig. 1). When severely bobbed significant. females of the genotype Xbb/XNo/w19 were mated to Magnification Occurs in Males with Incomplete Y Chromo- XNO/ Y males, no magnified Xbb chromosomes were found somes. Although it was not possible to test males carrying among 715 Xbb/X-No and Xbb/XNo/w19 female offspring only yL or Ys fragments, we could combine partial yL and Table 3. Magnification in males with two Y fragments and with Ybb- Xbb Score for bobbed phenotype Cross Chromosome B 99 BorBs 99 bb i99 bbm 99Y CT C % Magnification with w19 Ys and w28 yL Xbb/wl9/w28 cT X B/X-NO 9 9 y bb2 450 143 285 489 66.6 y UC03 544 164 313 484 65.6 Magnification with Ybb- Xbb/Ybb- cd X B/X-NO 9Y9 y bb2 202 80 69 180 46.3 y uco3 359 84 214 288 71.8 Magnifying males carrying either the wl9/w28 combination or Ybb- were mated to tester females heterozygous for the X-NO and an Xbb+ (B) chromosome. Progeny were classed as bb or bbm on the basis of posterior scutellar bristle length. Downloaded by guest on September 27, 2021 Genetics: Komma and Endow Proc. Natl. Acad. Sci. USA 84 (1987) 2385

YL YS * NO required in YY

requ i red in _ FIG. 2. Y required for magnification in female and male Drosophila. The region required in females is represented by the v24 X-Y translocation chromosome. The region required in males is represented by the w28 and w19 X-Y translocation chromosomes and includes the male fertility factors on yL and Ys.

partial Ys fragments in males and test the effect of such a about by this yL translocation chromosome. Therefore, it combination on magnification of an Xbb. Males of the seems unlikely that the requirement for a yL segment in genotype Xbb/w19/w28 are severely bobbed but fertile. As female magnification is a requirement for the yL collochore. shown in Table 3, this combination produces a high frequen- Furthermore, the collochores are thought not to be active in cy of bbm for two different Xbb chromosomes. The percent- female , and, although they are involved in pairing of age ofmagnification is comparable to that observed when the homologous chromosomes in male meiosis, their involve- same X chromosomes are magnified with Ybb-. When males ment in sister chromatid pairing is uncertain. of the Xbb/wl9/w28 genotype were mated individually, Finally, the data suggest that magnification requires an many produced more than one magnified offspring (cluster- interaction between a severely bobbed phenotype and Y ing). Clusters of magnified offspring from single males sug- chromosomal factor(s), probably on yL, and is not uniquely gest that some of the events leading to magnification occur induced by the Ybb- chromosome. We obtained magnifica- premeiotically (2, 13). Several females bearing an Xbbm tion in both males and females with Y fragments that have no chromosome from Xbb/wl9/w28 males were mated to ancestral relationship to the Ybb- chromosome. In view of X-No/Y tester males in order to look for evidence of the evidence that Ybb- is in some sense "constitutive" for instability. No reversions from bbm to bb were seen in any of magnification (13), we propose the following: The Ybb- the crosses, indicating that the Xbbm chromosomes were chromosome may create the conditions necessary for mag- stably magnified. nification in males that are phenotypically bbW. However, if the males are strongly bobbed in phenotype, then any Y DISCUSSION chromosome carrying the necessary region of yL will induce magnification. This model is consistent with the observation The data presented here are consistent with the hypothesis of magnification in combinations that do not include Ybb- that, in addition to a severely bobbed phenotype, magnifi- such as bb/ Ybb, "Vbb/O and X-No/Ybb (4, 13, 20-22). cation in female Drosophila requires some factor or factors The finding that sequences that may be involved in the located in the distal part of yL (Fig. 2). We examined five regulation of magnification reside on the Y chromosome is independently derived yL fragments and showed that they unexpected since the Y chromosome in Drosophila, as in promote magnification of an Xbb chromosome in females, other organisms, carries few genetic loci that have been whereas two independently derived Ys fragments that we identified besides the male fertility factors (16, 17, 23). The examined either do not magnify or do not result in magnifi- precise function of the Y chromosome in magnification, cation of an Xbb chromosome in females. In short, we do not whether in males or females, remains unknown. The fact that observe magnification in severely bobbed females that do not a relatively limited region ofyL, such as that contained within contain the distal part of yL. In addition, magnification does the v24 fragment, suffices to bring about magnification in not occur in females that are not severely bobbed, even when females suggests that a specific locus or loci are required. the entire Y chromosome including distal yL is present (5). Recombination between the X and Y chromosomes in We have no means at present ofdetermining whether or not Drosophila is a relatively rare event (19), unlike the obliga- the region ofthe Y chromosome required for magnification in tory recombination that occurs in the pseudoautosomal females is the same as that required for magnification in regions ofthe X and Y chromosomes in mice and humans (24, males. Separation of the factors necessary for male magni- 25). Work in mammalian systems provides a precedent for a fication from those needed for male fertility would be very high frequency of recombination within a localized area, difficult. It is possible, however, that some information which also appears to be characteristic ofthe sister chromatid regarding this question may be obtained by adding additional exchanges induced by magnifying conditions in Drosophila Y fragments to magnifying genotypes in order to learn males (3). If the Y region required for magnification encodes whether or not the additional Y chromosome material in- or regulates protein products required for recombination, creases the frequency ofmagnification. Such experiments are these products may mediate sister chromatid exchange, currently underway in both males and females. specificity for the ribosomal genes or inequality of sister Our data also demonstrate that magnification occurs readi- chromatid exchange. Another possibility is that the yL ly in males that are missing the section of the Y chromosome chromosome region is required for some form of gametic from the distal end of the nucleolus organizer through the selection, resulting, for instance, in a preferential recovery of centromere (Fig. 2). In addition to the nucleolus organizer, crossover gametes. This would help to explain the high this deleted region contains the Ys collochore or chromo- frequency with which magnification occurs in male Droso- some pairing site, which is active in male meiosis. We have phila. A final possibility is that the yL region may be needed not determined whether the region required for magnification for sister chromatid pairing, although our results indicate that in females includes the yL collochore. The yL collochore has the Ys collochore is not necessary for magnification to occur been localized only to the proximal half of yL (8) and may or in females. may not be proximal to the breakpoints of the w28, v31, or P7 translocation chromosomes. However, the breakpoint of We thank D. L. Lindsley for suggesting some of the experiments the v24 translocation chromosome is close to the tip of yL reported here and for providing appropriate stocks, K. C. Atwood (Fig. 1) and, therefore, is almost certainly distal to the yL for encouraging remarks, and S. A. Johnston and A. H. Yamamoto collochore. Magnification in females is nonetheless brought for helpful comments on the manuscript. This study was supported Downloaded by guest on September 27, 2021 2386 Genetics: Komma and Endow Proc. Nati. Acad. Sci. USA 84 (1987)

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