Proc. Natl. Acad. Sci. USA Vol. 91, pp. 3539-3543, April 1994 Genetics Insertional of Drosophila heterochromatin with single P elements

PING ZHANG AND ALLAN C. SPRADLING Howard Hughes Medical Institute Research Laboratories, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210 Contributed by Allan C. Spradling, December 28, 1993

ABSTRACT Insertional mutagenesis with transposable P efficiently generated throughout diverse regions of hetero- elements has greatly facilitated the identification and analysis chromatin by suppressing position effects genetically. of genes located throughout the 70% of the Drosophila mela- nogaster classified as euchromatin. In contrast, genet- ically marked P elements have only rarely been shown to MATERIALS AND METHODS transpose into heterochromatin. By carrying out single P Drosophila Stocks. Flies were grown on standard corn element insertional mutagenesis under conditions where posi- meal/agar media (1), at 220C. Unless stated otherwise, strains tion-effect variegation was suppressed, we efficiently generated and are as described in ref. 19. The same starting strains containing insertions at diverse sites within centromeric strain used previously (16) was employed for the screen in and Y-chromosome heterochromatin. The tendency of P ele- Fig. 1. It contains an X-linked insertion ofthe PZ transposon, ments to transpose locally was shown to operate within het- which carries the rosy+ (ry+) eye color gene. The isolation of erochromatin, and it further enhanced the recovery of hetero- 24 lines containing PZ insertions on the Y chromosome was chromatic insertions. Three of the insertions disrupted vital reported previously (16). The 95-2 strain was identified in a genes known to be present at low density in heterochromatin. screen for local transposition of the PZ element in the male Strains containing single insertions will greatly germ line (18). facilitate the structural and functional analysis of this poorly Genetic Analysis of Heterochromatic Insertions. General understood genomic component. information concerning single P element insertion screens was provided previously (14, 20). Briefly, the initial screen Like those ofmost eukaryotes, the chromosomes ofDrosoph- was carried out as follows (Fig. 1). F1 males of genotype ila melanogaster are divided into large, conspicuous euchro- X(PZ)/ Y; Sb A2-3/ry containing an X-linked PZ insertion and matic and heterochromatic domains (see Fig. 1) distinguished the A2-3 transposase source were generated to activate the by divergent structural and functional properties (1). Drosoph- element. Individual F1 males were crossed to C(1)RM/O; ila euchromatin accounts for approximately 70%o of the ge- ry/ry females in vials. F2 C(1)RM/ Y; ry/ry females carried a nome, and it can be subdivided by the detailed banding pattern Y chromosome to suppress position effect and were virgins in polytene chromosomes into approximately 5000 regions because their X/O male siblings were sterile due to the lack averaging 25 kb in length (2). In contrast, metaphase chromo- of a Y chromosome. A single F2 female bearing a new PZ some banding has resolved Drosophila heterochromatin into insertion as indicated by ry+ (i.e., wild type) eye color was about 60 regions averaging 1000 kb (3-5). collected from each vial and mated to X'Y/O; ry/ry males. Drosophila heterochromatin houses diverse genetic func- Candidate lines bearing heterochromatic insertions were tions, despite an abundance of satellite and repetitive DNAs identified by the failure of more than the expected 50% of F3 (6). More than 20 vital genes have been identified within progeny females to express ry+. Other lines were discarded autosomal heterochromatin (7, 8) and six Y chromosome and the sites of the insertions in the candidate lines were fertility factors are required exclusively during spermatogen- mapped to individual chromosomes and balanced (see be- esis (4, 9). Other heterochromatic regions interact with low). The screens involving Y95-2 were similar to the initial specific euchromatic genes (10, 11), while rearrangements screen except as follows. Transposition was activated in thatjuxtapose heterochromatin and euchromatin cause char- X/Y95-2; Sb A2-3/ry males or C(1)RM/Y95-2; Sb A2-3/TM6, acteristic local disruptions in gene expression known as ry females. Because insertions on neither the A2-3 nor the position-effect variegation (12, 13). Technical difficulties in TM6, ry chromosome were desired, no third chromosome analyzing heterochromatic regions rich in repetitive DNA insertions were retained from the screen in which the Y95-2 have limited our ability to systematically analyze these element was mobilized in females. phenomena. Insertions were balanced by scoring the meiotic segrega- Insertional mutagenesis with single P elements (14) has the tion of ry+ expression in those progeny where it was ex- potential to greatly facilitate studies of heterochromatin and pressed. In cases where expression was too weak, an extra its genes. However, previous attempts to insert genetically Y chromosome or PCR assays were used to determine marked P elements into heterochromatin have had only segregation. Southern blots were carried out on lines derived limited success. Insertions onto Dp1187, a well-defined from Y95-2 and bearing Y-linked insertions to verify that new 1300-kb minichromosome (15), were obtained only within a bands flanking the ends of the PZ element were present. small block of subtelomeric heterochromatin, not within a These tests revealed that some candidate strains derived from much larger region of centromeric heterochromatin (16-18). Y95-2 arose by internal Y chromosome rearrangement rather It remained uncertain whether genetically marked P elements than by transposition; these were not studied further. are simply unable to insert into centromeric heterochromatin Strains bearing lethal heterochromatic PZ insertions were or ifsuch insertions were not detected due to position effects. tested for genetic complementation with strains bearing de- We now report that single P element insertions can be ficiencies that uncover all the known heterochromatic vital genes. The strains used for CH(2)5 were as follows: Dfi2L)C' and the strains used for all The publication costs ofthis article were defrayed in part by page charge (21), Dft2L)ltxlO, Dft2L)lth2l (22); payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: DAPI, 4',6-diamidino-2-phenylindole. 3539 Downloaded by guest on October 2, 2021 3540 Genetics: Zhang and Spradling Proc. Natl. Acad Sci. USA 91 (1994) element insertions recovered previously in subtelomeric (15) 1\ i. or chromosome 4 (27, 28) heterochromatin all displayed position-effect variegation. Insertions lying deeper within heterochromatin might have been missed if marker gene expression had been completely suppressed. Such insertions i -XA hE I might be recovered under conditions where heterochromatic position effects were strongly suppressed by the presence of an extra Y chromosome. Furthermore, insertions subject to ./ t s 1 *a..f; ) ^ / :: )E strong position effect should be easily identified, since I ': marker gene expression from these sites would be lost or

V Y, / greatly reduced in a normal genetic background. V X To test this approach we carried out a single P element mutagenesis screen in which new transpositions of an V P. A X-linked P element marked with the rosy+ (by+) eye color gene (hereinafter called "PZ") were identified in flies bearing FiG. 1. Single P element insertional mutagenesis of heterochro- an extra Y chromosome (Fig. 1B; for further details, see matin. (A) Schematic drawings of the Drosophila melanogaster Materials andMethods). When flies from 4% individual lines chromosomes illustrate the location and size of heterochromatin bearing new independent PZ transpositions were crossed to (stippled boxes) and euchromatin (open boxes). Centromeres are remove the extra Y chromosome, 10 strains displayed a ry- represented as small circles. The positions ofthe starting P elements X(PZ) and Y95-2 are shown. (B) Crossing scheme for generating eye color (Table 1). The relatively high frequency of these Y-dependent insertions. For details and genetic nomenclature see "Y-dependent" insertions (10/4% = 2%6) compared with the Materials and Methods. rate of heterochromatic insertions in a previous study (24/ 7825 = 0.3%) (16) encouraged further studies. CH(3) strains were as follows: Df(3L)2-30, Df(3L)9-56, A Genetically Silent P Elment Insertion Within Y Chromo- Dft3L)2-66, D~ft3L)1-166, Dff3L)8A-80, Dff3L)6-61, Dff3L)3- some Heteroebromatin. We next characterized line 95-2, 52, Df(3L)1-16, Dff3LR)10-26, Df(3LR)6B-29, Dff3R)10-65, generated during previous experiments to investigate local P and Dfl3R)4-7S (8). Complementation was observed in all element transposition (18). In addition to a euchromatic crosses except as noted in the text. insertion, 9S-2 males were found by Southern blotting to Reversion tests by PZ excision were carried out essentially contain a second PZ insertion on their Y chromosome that as described (14). One hundred independent lines that failed was separated into a derivative line called Y95-2. Fig. 2 shows to express ry+ were generated from each strain and tested for that a single band complementary to the P element probe was viability in combination with the starting insertion. (A few of observed in strain Y95-2 DNA from males but not females as these derivatives still retained the original element but were expected for a Y-linked insertion. Surprisingly, the Y95-2 phenotypically ry- due to position effect.) In the case of insertion appeared to be structurally normal but failed to strains CH(2)S and CH(3)7, >50% of ry derivatives were viable when heterozygous with the starting chromosome; the express ry+ as judged by eye color, even in the presence of other lines produced no viable derivatives. an extra Y chromosome to suppress position-effect variega- DNA Probes. Plasmid pMC1871 (23) and Carnegie 20 (24) tion. Full ry+ pigmentation was restored when the Y9S-2 PZ DNA, which together contain 12 kb ofthe 14-kb PZ element, element was remobilized to euchromatic sites, proving that a were used to label insertions in situ. Unique sequence strong heterochromatic position effect suppressed ry+ ex- genomic DNA cloned from cytogenetic positions 24C or 100C pression at the Y95-2 insertion site. These observations (L. Schneider and A.C.S., unpublished work) were used to identified an insertion site where ry+ expression was subject mark chromosome 2L and 3R, respectively. Probes used for to position effects that could not be reversed by an extra Y Southern hybridization were described previously (18). chromosome but where the element's capacity to undergo In Situ Hybrtion to Metaphase Chromosomes. Fluores- transposition was little affected. cence in situ hybridization (Chromosome In Situ Hybridiza- An Efficient Screen to Recover Heterochromatic Insertions. tion System, Oncor) was carried out as described (25). Brains The unusual properties of the Y95-2 line allowed a more were dissected from third-instar larvae and squashed as efficient screen for heterochromatic insertions to be carried described (3), but no pretreatments to accumulate cells in out. The screen was similar to the previous one, but the Y95-2 metaphase or staining was used. DNA was labeled with insertion served as the starting site. This scheme appeared biotinylated dATP by nick translation (BioNick kit, BRL). It likely to provide several major advantages. Because flies is difficult to localize sequences within the heterochromatin bearing the Y95-2 insertion were phenotypically ry-, progeny of the small Drosophila chromosomes when staining and flies were expected to acquire a wild-type eye color only banding are carried out prior to in situ hybridization (26). when the PZ element transposed to a new and less suppres- Consequently, we developed methods that allowed bands to sive chromosomal site, facilitating the identification oftrans- be visualized on the labeled chromosomes. Prior to mounting position events. Starting with a genetically silent Y-linked in propidium iodide/antifade (Oncor), slides were stained for insertion was also expected to generate many new Y chro- 5 min with 4',6-diamidino-2-phenylindole (DAPI) at 0.5 pig/ mosome insertions, because P elements jump locally with ml. Each field was photographed by using a Zeiss Axiophot increased frequency (17, 18). Finally, transpositions were microscope and Elite400 film (Kodak) both in the DAPI channel and using filters to record both the signal and Table 1. Summary of mutagenesis screens propidium fluorescence. The film was digitized and chromo- Heterochromatic somes in each field were aligned and analyzed by using Starting Total Euchromatic insertions Adobe Photoshop on an Apple Quadra computer and were position* insertions insertions Y-linked Autosomal printed with a Kodak printer. X 496 486 (98%) 3 (0.6%) 7 (1.4%) Y9S-2M 1 134 (94%) 4 (2.8%) 5 (3.5%) RESULTS Y95-2F 158 145 (92%) 8 (5%) 5 (3.2%) Recovery of Heterochromatic Insertions Under Conditions *Transpositions recovered from males (Y95-2M) and females (Y95- Where Position-Effect Variegation Is Suppressed. Single P 2F). Downloaded by guest on October 2, 2021 Genetics: Zhang and Spradling Proc. Natl. Acad. Sci. USA 91 (1994) 3541 AX B Mapping Insertions Within Heterochromatin. To determine -2 if the insertions in strains with Y-dependent expression _ -J ry+ .- _J e; were located in heterochromatin, we mapped their positions : in the genome. First the insertion in each new line was genetically mapped to a chromosome and was balanced to learn if it was associated with any recessive phenotypic effects. Fifteen lines mapped to the Ychromosome, while the _ . _~ remaining 17 were divided between the two major autosomes. The chromosomes bearing insertions in 5 of the 17 lines with ...., autosomal insertions caused lethality when homozygous. The position ofthe PZ element was then mapped in greater C detail, using in situ hybridization. Since all the insertions R R R R were found to lie within heterochromatin it was not possible to utilize salivary gland chromosomes for these experiments. P P PvC e vV Heterochromatic regions are severely underrepresented in polytene chromosomes, rendering them unsuitable for gene

8 4 Kt 4 t

FIG. 3. Localization of Y-dependent PZ insertions by in situ hybridization to mitotic chromosomes. Each panel shows aligned composite images of the chromosome containing the insertion labeled with DAPI (blue) or propidium iodide (red). The hybridization signals for the PZ element (arrow) and two marker genes (2L or 3R) appear as yellow spots. Since a fraction of the PZ probe consists of By+ gene sequences, cross-hybridization to this locus on chromosome 3R was frequently observed (ry). Our interpretation of the major heterochromatic bands (hl-h58) revealed by DAPI staining (ref. 19; see also Fig. 4) are indicated below the DAPI image. The strains shown are as follows: Yc8 (A), Y512 (B), Y121 (C), CH(2)423 (D), CH(2)11 (E), CH(2)8 (F), CH(3)4 (G), CH(3)7 (H), and CH(3)148 (I). Downloaded by guest on October 2, 2021 3542 Genetics: Zhang and Spradling Proc. Natl. Acad. Sci. USA 91 (1994) Chromosome Y Insertions: with euchromatin, the PZ elements in lines CH(2)6, 1 3 5 7 9 11 13 15 17 19 21 23 25A CH(2)470, CH(3)J, CH(3)148, and CH(3)336 had integrated 2 4 6 8 10 12 14 16 18 20 22 24 25B deep within these centric regions. Several Insertions Inactivate Heterochromatic Genes. The C 10,16 95-2 five strains bearing lethal mutations (Table 2) were tested c2, c3, 1 genetically to determine if the lesions were caused by their P 364 4 13,17 c8 insertions (Materials and Methods). Failure to complement a 302 'C Chromosome 2 Insertions: specific heterochromatic deletion or reversion after P ele- ment excision was taken as strong evidence that the insertion 2L 35 36 37 38 39 4041 42 43 44 45 46 2R was responsible for the lethality. Strains CH(3)1 and all C CH(3)148 complemented available deficiencies, but 5,8 6 strains CH(3)4 and CH(3)7 both failed to complement the 470 423 1, 11 overlapping deficiencies Df(3L)3-52 and Df(3L)1-16. Exci- sion ofthe P element in strain CH(3)7 frequently reverted the Chromosome 3 Insertions: lethality associated with the mutant chromosome, showing 47 48 49 50 51 52 53 54 55 56 57 58 K 3R that the P element was responsible for the lethal . CH(3)4 did not revert in similar tests; however, this was - C- 4, 7 148 1 probably due to the presence of a "background" lethal _5 336 mutation located elsewhere on the chromosome, since it 6 9,10 failed to complement CH(3)1. Thus the lethality in at least two of the four chromosome 3 lines was associated with their FIG. 4. Summary of single P element integration sites with PZ insertions. It remains possible that the P elements in respect to cytogenetic banding. Diagrams of the major heterochro- CH(3)1 and CH(3)148 also cause lethal mutations located matic bands (labeled 1-58 above the idiograms) on chromosomes Y, outside the available deficiencies but were isolated on chro- 2, and 3 are shown (19). Below each diagram are bars indicating the regions to which the insertions were mapped in the Y-dependent lines mosomes containing a background lethal mutation. (see Table 2). The approximate brightness expected of particular A Vital Locus Located in Proximal 2L Heterochromatin. chromosome regions after DAPI staining is indicated by the degree Line CH(2)5 reverted to viability after excision of the P of shading in the diagram (black = bright fluorescence, white = dim element, but it complemented Df(2L)C', which uncovers all fluorescence). Only the heterochromatic regions of the chromo- the previously known lethal complementation groups in 2L somes are shown; dotted lines indicate the basal regions of the left heterochromatin (7). Therefore, CH(2)5 defines a previously (L) and right (R) euchromatic arms. undescribed vital locus in the bands h36-37, proximal to the previously known genes (5). In contrast, all 24 insertions recovered without suppressing position-effect variegation mapped exclusively within the DISCUSSION subtelomeric regions hl-3 or h24-25 (data not shown). Many Suppressing Position-Effect Variegation Increases the Re- of the lines derived from the Y95-2 element appeared to have covery of P Element Insertions Within Heterochromatin. Our integrated locally near its site of insertion in hll-13. Auto- experiments showed that position effects limit the recovery somal insertions were located at multiple sites within the of genetically marked P elements within heterochromatin by centromeric heterochromatin ofchromosomes 2 and 3 (Table extinguishing marker gene expression. No PZ insertions in 2 and Fig. 4). Genetically marked P elements had not autosomal heterochromatin had been reported previously, to previously been characterized within autosomal heterochro- our knowledge. Only about 0.33% ofPZ transpositions were matin. Although some were located fairly close to junctions recovered on the Y chromosome in the absence of suppres- Table 2. Heterochromatic P element insertions Y-linked Autosomal New Starting New Starting Pheno- Strain site site* Strain site site* typet Y95-2 hll-13 CH(2)1 h44-46 Y95-2F v Y2 hll-13 Y95-2Fr CH(2)5 h36-37 Y95-2F I Y4 h2-4 Y95-2Fe CH(2)6 h41-44 Y95-2F v Y10 hl-3 Y95-2Fr CH(2)8 h36-37 Y95-2F v Y12 hll-13 Y95-2F,r CH(2)11 h44-46 Y95-2F v Y13 hll-13 Y95-2F,r CH(2)423 h42-43 Y95-2M v Y16 hl-3 Y95-2Fe CH(2)470 h37 Y95-2M v Y17 hll-13 Y95-2Fe CH(3)1 h55-56 X I Yc2 hl-3 X CH(3)4 h47-48 X 1 Yc3 hl-3 X CH(3)5 h47-49 X v Yc8 h15-16 X CH(3)6 h47 X v Y121 h8-9 Y95-2M,r CH(3)7 h47-48 X I Y302 hll-13 Y95-2Mr CH(3)9 h48-50 X v Y364 hl Y95-2M,e CH(3)10 h48-50 X v Y512 h14-15 Y95-2Me CH(3)148 h49-50 Y95-2M v CH(3)309 ND Y95-2M v CH(3)336 h54-57 Y95-2M v ND = not determined. *PZ transposon starting sites: X = X chromosome; Y95-2M = Y chromosome in male; Y95-2F = Y chromosome in female; r and e = starting PZ transposon retained (r) or excised (e). tl = lethal; v = viable. Downloaded by guest on October 2, 2021 Genetics: Zhang and Spradling Proc. Natl. Acad. Sci. USA 91 (1994) 3543 sion (16), compared with 2.0o when variegation was sup- Insertional Mutagenesis and Large-Scale Structural Studies pressed (Table 1), a significant increase (P < 0.01). The ry+ of Heterochromatin. Many of the fascinating properties as- marker gene present on all the Y-linked insertions showed sociated with heterochromatic regions are unlikely to be strong position-effect variegation regardless ofhow the strain understood until the physical structure and sequence ofthese had been isolated. The failure to recover insertions refractory genomic regions can be determined. The Dp1187 minichro- to the position effect indicates that virtually all sites of P mosome provides one such model (16). Single P element insertion within heterochromatin are associated with strong insertions greatly assisted the structural analysis of a 10-kb position effects. Conversely, all the insertions we selected as segment of subtelomeric heterochromatin on this chromo- responding to a modifier of position-effect variegation were some (16). Our experiments suggest that reiteration of the screen we described would generate a large number of found to reside in heterochromatin. additional novel insertion sites throughout heterochromatin The fact that insertions such as Y95-2 would not have been generally. The ability to generate such insertions now prom- recovered in the screens described here indicates that the ises to open up many new genomic areas to detailed molec- presence ofan extra Ychromosome is insufficient to suppress ular and genetic analysis. the position effects associated with some heterochromatic sites. There are several ways an even greater level of sup- We thank Ms. Anita Hawkins and Dr. Constance Griffin for pression might be achieved. Position-effect variegation is teaching us to carry out in situ hybridization to metaphase chromo- somes. Deficiency stocks were provided by Dr. Barbara Wakimoto reduced at high temperatures and in the presence of numer- and by the Bloomington Stock Center. This work was supported in ous dominant modifiers (13). Wild-type pigmentation was part by U.S. Public Health Service Grant GM27875 and by the partially restored to Y95-2 flies that carried an extra Y Howard Hughes Medical Institute. chromosome and were reared at 270C (data not shown). It is 1. Ashburner, M. (1990) Drosophila: A Laboratory Handbook likely that an even wider distribution of heterochromatic (Cold Spring Harbor Lab. Press, Plainview, NY). insertions could be recovered in screens patterned after those 2. Lefevre, G. (1976) in The Genetics and Biology ofDrosophila, described here but which use different modifiers or a com- eds. Ashburner, M. & Novitski, E. (Academic, New York), pp. bination of factors to suppress position-effect variegation. 32-64. P Elements Preferentially Transpose Locally on the Y Chro- 3. Gatti, M. & Pimpinelli, S. (1983) Chromosoma 88, 349-373. 4. Bonaccorsi, S., Pisano, C., Puoti, F. & Gatti, M. (1988) mosome. Transposition is enhanced as much as 50-fold near Genetics 120, 1015-1034. the starting sites ofP elements located within euchromatin or 5. Dimitri, P. (1991) Genetics 127, 553-564. subtelomeric heterochromatin (17, 18). Local transposition 6. Gatti, M. & Pimpinelli, S. (1992) Annu. Rev. Genet. 26, probably caused the significant increase (P < 0.05) in new 239-275. Y-linked insertions we recovered from the Y95-2 starting site 7. Hilliker, A. (1976) Genetics 83, 765-782. compared with the X-linked site (4.3% vs. 0.6%). As ob- 8. Marchant, G. E. & Holm, D. G. (1988) Genetics 120, 519-532. 9. Brosseau, G. E. (1960) Genetics 45, 257-274. served previously (18), more putative local insertions were 10. Parry, D. & Sandler, L. (1974) Genetics 77, 535-539. recovered in the female germ line than from males (5.0% vs. 11. Livak, K. J. (1984) Genetics 107, 611-634. 2.8%); however, a larger sample would be required to prove 12. Henikoff, S. (1990) Trends Genet. 6, 422-426. that this difference was significant. 13. Reuter, G. & Spierer, P. (1992) Bioessays 14, 605-610. Insertional Mutagenesis Will Facilitate the Cloning and 14. Cooley, L., Kelley, R. & Spradling, A. C. (1988) Science 239, Analysis of Heterochromatic Genes. More than 20 genes have 1121-1128. 15. Karpen, G. H. & Spradling, A. C. (1990) Cell 63, 97-107. been genetically defined within autosomal heterochromatin 16. Karpen, G. H. & Spradling, A. C. (1992) Genetics 132, 737- (7, 8). At least some of these "heterochromatic" genes 752. undergo variegated position effects when relocated into eu- 17. Tower, J., Karpen, G., Craig, N. & Spradling, A. C. (1993) chromatin (22, 29, 30), suggesting that they may require a Genetics 133, 347-359. heterochromatic location for normal function. Single P ele- 18. Zhang, P. & Spradling, A. C. (1993) Genetics 133, 361-373. ment insertions that disrupt heterochromatic genes will make 19. Lindsley, D. L. & Zimm, G. L. (1992) The Genome of Dro- them much easier to clone and analyze, and they will make sophila melanogaster (Academic, New York). it easier to study the mechanism 20. Berg, C. A. & Spradling, A. C. (1991) Genetics 127, 515-524. of "reverse" position 21. Hilliker, A. & Holm, D. G. (1975) Genetics 81, 705-721. effects. Heterochromatic genes have previously been mu- 22. Wakimoto, B. T. & Hearn, M. G. (1990) Genetics 125,141-154. tated and cloned only by using small unmarked P elements 23. Casadaban, M., Martinez-Arias, A., Shapira, S. & Chou, J. mobilized by hybrid dysgenesis (31-34). (1983) Methods Enzymol. 100, 293-308. Single P insertions will make it easier to map existing 24. Rubin, G. M. & Spradling, A. C. (1983) NucleicAcids Res. 11, heterochromatic genes and to identify new ones. A series of 6341-6351. lethal single P lines whose insertions were cytogenetically 25. Hawkins, A. L., Jones, R. L., Zenbauer, B. A., Zicha, M. S., localized at intervals throughout centromeric heterochroma- Collector, M. J., Sharkis, S. J. & Griffin, C. A. (1991) Cancer tin would help in determining which regions are absent from Genet. Cytogenet. 64, 145-148. 26. Lohe, A. R., Hilliker, A. J. & Roberts, P. A. (1993) Genetics existing heterochromatic deficiencies. Furthermore, a series 134, 1149-1174. of simple deletions could be generated from these lines by 27. Spradling, A. C. & Rubin, G. M. (1983) Cell 34, 47-57. imprecise P element excision. Our results demonstrated that 28. Clark, S. H. & Chovnick, A. (1986) Genetics 114, 819-840. new genes will be identified directly by insertional mutagen- 29. Hearn, M. G., Hedrick, A., Grigliatti, T. A. & Wakimoto, esis. Although the density of genes in autosomal heterochro- B. T. (1991) Genetics 128, 785-797. matin is thought to be less than 1% as great as in euchromatin, 30. Eberl, D. F., Duyf, B. J. & Hilliker, A. J. (1993) Genetics 134, mutations were recovered at about the same rate: 17% (3/17) 277-292. vs. 10% (35). Perhaps genes constitute a high fraction of the 31. Devlin, R. H., Bingham, B. & Wakimoto, B. T. (1990) Genetics available target sites within heterochromatin. Previous stud- 125, 129-140. ies had failed to 32. Parks, S. & Wieschaus, E. (1991) Cell 64, 447-458. mutate any genes within bands h36-38, which 33. Orenic, T. V., Slusarski, D. C., Kroll, K. L. & Holmgren, make up 70% of 2L heterochromatin (5). We showed that R. A. (1990) Genes Dev. 4, 1053-1067. CH(2)5 identified such a gene. Thus the clustering of muta- 34. Mitchelson, A., Simonelig, M., Williams, C. & O'Hare, K. tions in h35 observed previously (5) may have been due to the (1993) Genes Dev. 7, 241-249. lack of appropriate means to recover mutations in more 35. Cooley, L., Berg, C. & Spradling, A. C. (1988) Trends Genet. proximal loci. 4, 254-258. Downloaded by guest on October 2, 2021