Genetic Analysis of the Heterochromatin of Chromosome 3

Home , Chromosome 3

Genetic Analysisof the Heterochromatin of Chromosome 3 in Drosophila melanogaster. I. Products of Compound-Autosome Detachment

Gary E. Marchant and David G. Holm Department of Zoology, The Universityof British Columbia, Vancouver, British Columbia, Canada V6T 2A9 Manuscript received September 15, 1987 Revised copy accepted July 1, 1988

ABSTRACT The heterochromatin of the third chromosome is the largest uncharacterized region of the Drosophila melanogaster genome, and the last major block of D. melanogaster heterochromatin to be thoroughly analyzed. In the present study, this region was genetically dissected by generating and analyzing a series of attached, detached and reattached third chromosomes. Separate detachment experiments were conducted for all 12 possible combinations of four newly synthesized sister-strand compound-31 and three newly synthesized sister-strand compound-3R chromosomes.A total of 443 recessive lethal detachment products carrying putative heterochromatic deficiencies were tested for complementation in a several-stage complementation analysis. The results revealed the presence of seven separable vital regions in the heterochromatin of chromosome 3. Attempts to reattach deficiency-carrying detachment products established that six of these vital regions are on the left arm, but only one is on the right arm. An analysis of the types and frequencies of detachment-product deficiencies generated in each detachment experiment permitted the genetic characterization of the progenitor compounds. It was also possible to determine the proximal-distal orientation of the genes on each arm, and to identify possiblebreakpoints for each lethal detachment product produced. The results of this study suggestthat vital genes in the heterochromatin of the third chromosome are not randomly distributed between, nor within, the heterochromatic blocks of the left and right arms.

HROMOSOME 3 of Drosophila melanogastercon- that the constitutive heterochromatin of D. melano- C tains the last major uncharted block of hetero- gaster and other organisms is almostentirely com- chromatin to be genetically analyzed in this species. posed of long, homogeneousblocks of individual sat- Heterochromatin is of two types, facultative and con- ellite DNA sequences tandemly arranged in a chro- stitutive(BROWN 1966), but only constitutive is mosome-specific order. The estimatedamount of present in the chromosomes of Drosophila. Unlike the satellite DNA in eachblock of heterochromatin almost euchromatic regionsof the genome, heterochromatin completelyaccounts for the total amount of DNA remains condensed and darkly stained throughout thepresent (PEACOCKet al. 1977b), suggesting that few, cell cycle of most cells. It occupies identical positions if any, othertypes of DNA sequences reside withinD. on homologous chromosomes and in Drosophila ac- melanogaster heterochromatin.This view was sup- counts for the proximal half of the X chromosome, ported by the discovery of junction DNA molecules the entire Y, the proximal 1/5 to 1/4 of the arms on that covalently link adjacent blocks of satellite DNA the two major autosomes, and ofmost the small fourth (BRUTLAGet al. 1977).While a few nonsatellite se- chromosome.In total, heterochromatin represents quences appear to be in heterochromatin, only trace approximately 28% of the total Drosophila genome amounts of satellite DNA are found outsidethe het- (PEACOCKet al. 1973). erochromatic regions (PEACOCKet al. 1977a; COHEN There is a striking correlation between regions of and BOWMAN1979). thegenome withlittle or nogenetic activity and Even though the molecular and structural proper- regions of heterochromatin. The DNA composition tiesof heterochromatin indicate a paucity if not a of constitutive heterochromatin wouldalso argue that complete absence of gene expression, a number of little genetic activity couldoccur in these regions. genetic functions have been mapped to the hetero- Constitutive heterochromatin is greatly enriched in chromatin ofD. melanogaster (see reviewby HILLIKER, satellite DNA, which is composed of very short nu- APPELS and SCHALET1980). Thenucleolus organizer cleotide sequences present in hundreds of thousands regions in both theX and Y heterochromatin contain of copies per genome (YASMINEH and YUNIS 1969; the tandemly repeated genes for rRNA(RITOSSA and RAE 1970;JONES and ROBERTSON1970; PARDUEand SPIEGELMAN1965). Although these genes are located GALL 1970;PEACOCK et al. 1973; GALL andATHER- at the large secondary constrictions in the heterochro- TON 1974). PEACOCKet al. (1973, 1977a) have shown matin, they retain many of the properties of hetero-

Genetics 120 503-517 (October, 1988) 504 G. E. MarchantG. and D. Holm chromatin when separated from the heterochromatic of recessive lethal and visible mutations allelic to the region (HILLIKER andAPPELS 1982). Male fertility in deficiencies of the heterochromatic region of chro- D. melanogaster requires the presence of six fertility mosome 2 revealed the presence of 13 nonrepetitive factors on the heterochromatic Y chromosome genes, seven to the left and six to the right of the (BROUSSEAU1960; KENNISON198 1; HAZELRIGG,FOR- centromere (HILLIKER1976). Cytological examina- NIT1 and KAUFMAN 1982; GATTI and PIMPINELLI tions of the detachment-product deficiencies in chro- 1983). Each fertility factor is involvedin a specific mosome 2 indeed demonstrated that the deficiencies step in sperm development (HARDY,TOKOYASU and were restricted to the heterochromatic regions. LINDSLEY1981) and seems to code for a different However, the euchromatin-like genes shown to be protein required for sperm development and fertility in chromosome 2 heterochromatin do not seem to (GOLDSTEIN,HARDY andLINDSLEY 1982). The recov- exist in the heterochromatin of the X or Y chromo- ery of EMS-induced (WILLIAMSON1970, 1972) and some (LINDSLEY, EDINGTONand VON HALLE1960; temperature-sensitive (AYLESet al. 1973) mutations SCHALETand LEFEVRE1973; HILLIKERand APPELS of male fertility factors suggest that they are single- 1982; KENNISON1981; GATTIand PIMPINELLI1983) copy, transcribed genes. However, on the basis of an (see also review by HILLIKER,APPELS and SCHALET extensive break-point analysis, GATTIand PIMPINELLI 1980). These findings raise the possibility that heter- (1983) havesuggested that at leastsome of the Y ochromatin in D. melanogaster may be heterogeneous fertility factors may have an enormous physical size, with respect to genetic activity. Sincethe heterochro- each containing up to several thousand kilobases of matin of the sex chromosomes has a very different DNA. In addition to the six fertility factors there is a genetic composition than the heterochromatin of at specific segment on the long arm of the Y required least one of the major autosomes, it would be of for normal sperm development. Deletion of this seg- significant interest to determine if the genetic content ment results in crystal formation in primary sperma- of chromosome 3, the other major autosome, con- tocytes, but not sterility (HARDYet at. 1984; LIVAK forms to this dichotomous pattern. The results of 1984). BALDWINand SUZUKI(197 1) which introduced the Other genetic properties have been mapped to spe- technique of generating proximaldeficiencies cific regions of D. melanogaster heterochromatin, al- throughthe detachment of compound autosomes, though it is not clear whether transcribed genes are suggested that chromosome 3 heterochromatin does involved. The chromosomal regions responsible for contain essential genes. However, there was insuffi- nucleolar dominance (DURICAand KRIDER 1978) and cient information about the number, distribution, re- compensationresponse (PROCUNIERand TARTOF petitiveness and nature of genes in chromosome 3 1978) are located in the X chromosome heterochro- heterochromatin to permit a validcomparison be- matin. A segment of the X heterochromatin also in- tween the heterochromatic gene content of the two teracts with a family of recessive maternal mutations major autosomes. including abnormal oocyte (SANDLER1979,1972,1977; In the present study we analyzed over 440 recon- PARRY andSANDLER 1974; PIMPINELLIet al. 1985). stituted third chromosomes generated through the Extra copies of a specific segment of X heterochro- radiation-induced detachment of 12 different combi- matin as well as two segments of the Y, can partially nations of C(3L)and C(3R).These compound auto- compensate for the maternal mutant defect in abnor- somes were newly generated as sister-chromatid at- mal oocyte progeny (SANDLER1970; PIMPINELLIet al. tachments from a recently established isogenic-3strain 1985). Finally, elements involved in the segregation to avoid the accumulation of recessive lethals, espe- distortion (SD) phenomenon have been mapped to cially near the centromere where the frequency of the heterochromatin of chromosome 2 (GANETSKY homozygosity is verylow (PARKER1954). Some of 1977; BRITTNACHERand GANETSKY1984; SHARP, these compounds carried duplications for the proxi- HILLIKERand HOLM1985), and recently MCKEEand mal heterochromatin on the opposite arm and some LINDSLEY987) (1 report that theheterochromatic sites were heterozygous for deficiencies. Although these on the X responsible for sex chromosome pairing are properties were not known prior to recovering and veryclosely linked and probably identical with the analyzing the products of detachment, they provided sites required for normal sperm development and for the essential genetic complexity for producing polar, fertility in the presence of duplicated Ys. nonpolar and centromere-spanning deficiencies that Proximaldeficiencies generated throughthe de- uncovered vital genetic loci in the heterochromatin tachment of compound-2 chromosomesin females of chromosome 3. Unlike the studies on chromosome revealed the presence of at least six genetic loci within 2 (HILLIKERand HOLM1975) where visible genetic the centromeric heterochromatin of chromosome 2 markers were uncovered by deficiencies to the left or (HILLIKERand HOLM1975). Further analysis of this to the right of the centromere, no genetic markers region by the induction with ethyl methane-sulfonate had been previously identified within the heterochro- Detachment Products of Compound 3 505 matic regions of chromosome 3. Consequently, the ment experiment was performed for each of twelve lines construction of sister-chromatid compound-3 chro- carrying a different combination of new C(3L) and C(3R) chromosomes. In each experiment, 2000 virgin compound- mosomes was attempted with representative detach- 3 females were treated with 2200 rad of gamma radiation ment products carrying deficiencies. This approach and mated in groups of fifty to TM3/Ly males in half-pint made it possible to assign each of the newly discovered bottles. The parents were cleared after 10 days, and off- genetic loci either to the right or to the left of the spring were collected at 18" for the next 20 days. Virgin centromere. females and males carrying detachment products recovered over TM3, and males carrying detachment products re- The procedures described above have made possi- covered over Ly chromosome, were established in stockover ble an extensive genetic investigation of the proximal TM3. heterochromatin of chromosome 3 confirming and Lethality tests: Each detachment product was tested for extending the original findings of BALDWINand SU- homozygous lethality by mating males and females carrying the same detached ZUKI (1971). The results of this study reveal that at ri Pp chromosome balanced over TM3. All lethality tests were carried out in duplicate in shell vials least six vital genetic loci occupy sites in the hetero- with two pairs of flies per vial. Recessive lethals were iden- chromatin to the left of the centromere and that at tified as the total absence of ri p@progeny. If the detachment least one locus is in the heterochromatin to the right. product was homozygous viable, then ri PpITM3 heterozy- gotes and homozygous ri Pp flies were expected in approxi- MATERIALS AND METHODS mately a 2:l ratio. All offspring in each vial were counted to ensure that semilethals would be detected. Detachment Genetic markers, rearranged chromosomes andexper- products carrying recessive lethals were maintained in stock imental conditions: Four standard and two compound-3 over TM3. Approximately 150 nonlethal detachment prod- strains of D. melanogaster were used in this study. All newly ucts were also saved. generated compound third chromosomes and their detach- Complementation tests: To make possible the analysis of ment products carried the proximal recessive markers radius a large number of detachment products without requiring incompletus (ri) on the left arm and pink-peach (Pp))on the an inordinate number of crosses, complementation testing right. Detachment products were balanced over was done in severalstages. First, lethal detachment products Zn(3LR)TM3, ri Pp sep Sb bx3* e' Ser (hereafter referred to from five of the 12 detachment experiments were selected as TM3), which hasbeen maintained in a stock heterozygous for complementation analysis. For each of these five sets all for the dominant mutation Ly. The proximal position of inter se crosses were tested for lethality. From these tests, a putative deficiencies was confirmed by crossover analysis separate complementation map was produced for each of using the two genetically marked third chromosomes st in the five sets ofdetachment chromosomes. ri eg and st eg Ki or a derivative. Two compound-3 strains, The second stage of the complementation analysis was to C(3L)VTl,se; C(3R)VKl,eSand C(3L)VH3,st; C(3R)SHl9, +, test between complementation groups of the five detach- were used to rescue the newly generated compound auto- ment experiments. Several detachment products were se- somes. Further information on these mutations and rear- lected from every complementation group in each of the ranged chromosomes can be obtained from LINDSLEYand five experiments. These chromosomes were then pooled GRELL(1968). Unless otherwise stated, all experimental and tested in all inter se combinations, producing an accu- crosses were carried out at 24 f 1 ' on standard Drosophila mulated complementation map for the five original experi- medium prepared from cornmeal, yeast, sucrose, dextrose ments. Further complementation tests were carriedout and agar with propionic acid added as a mold inhibitor. within complementation groups on the accumulated map Synthesis of new compound3 chromosomes: All com- wherever necessary to resolve any ambiguities. pound autosomes used for detachments were recovered as In the third stage of the complementation analysis tests sister-chromatid attachments generated from a strain iso- were carried out with all lethal detachment products from genic for the ri Pp third chromosome. This isogenic ri Pp the seven remaining experiments against a tester chromo- strain was obtained by mating a single ri p/TM3 male to some from each complementation group on the accumu- TM3/Ly females, and then mating siblings of the ri Pp/TM3 lated map. From the results of these crosses, all the detach- progeny to isolate a homozygous ri Pp stock. ri Pp males were ment products of the seven remaining experiments were collected and mated to virgin TM3/Ly females. Approxi- assigned to a complementation group on the overall map. mately 1500 virgin F1ri Pp/TM3 females were collected and Finally,some random complementation tests were con- treated with 2000 rad of y-radiation from a 6oCosource. ducted within each complementation group to ensure that The irradiated females were mated in groups of 25 to none of the complementation groups could be further sub- C(3L)VTl,se; C(3R)VKl,eSmales in half-pint milk bottles. divided. The parents were cleared after 10 days. New compounds In each stage of the complementation analysis described generated in females heterozygous for TM3 could be formed above, all tests involved crossing a pair of males carrying only through the attachment of sister chromatids of the one lethal balanced over TM3 to a pair of females carrying noninverted ri Pp homologue. The few surviving progeny a second lethal over TM3. All crosses were carried out in either carried a newly formed compound third, or arose as shell vials. Reciprocalcrosses were performedfor most a product of a nondisjunctional event. All ri e" offspring complementation tests.Crosses between two noncomple- carried a newly formed sister-strand C(3L) chromosome, menting lethals produced only ri pP/TM3 progeny. In crosses while se Pp individuals carrieda new sister-strand C(3R) between complementing lethals, ri Pp/TM3 and ri Pp/ri pP chromosome. Separate stocks were established for each pos- progeny were recovered in an approximate ratio of 2:1. sible combination of newly synthesized compound thirds. Recombination mapping:The proximal position of pu- All compound thirds generated for this study have been tative deletions carried by the detachment products was labeled with the letters VM followed by a number specifying confirmed by crossover analysis. A single detachment chro- the compound in the series. mosome from each complementation class was tested in two Recovery of detachment products: A separate detach- recombination mapping experiments. The first crossover 506 G. E. Marchant and D. G. Holm analysis determined if the deficiency on the tester chromosomes was to the left or to the right of eagle, the most cba def cba def proximal visible marker on the left arm of chromosome 3. Females heterozygous for a ri Pp detachment product over cbq def cbat def the marker chromosome st eg Ki were mated to homozygous st in ri eg males. F1 offspring were screened for crossovers between ri at map position47.0 and eg at map position 47.3. Recombinant progeny with a scarlet phenotype carried the heterochromatic region of the detachment chromosome, the reciprocal ri eg Ki product did not. Both classesof recombinant products were tested for lethality over the original detachment product. If the recombinant chromosome recovered in the scarlet progeny failed to survive over the detachment product, then the deficiency carried by the detachment product was to the right of the ri-eg region. Conversely, if the reciprocal ri eg Ki product failed to survive, the deficiency was in the left arm euchromatin. A comparable experiment was conducted for the right arm. Females carrying the detachment product over the cba del marker chromosome eg Ki were mated to homozygous ri Pp males.Crossovers in the region between Kinked at map position 47.6 and pink-peach at map position 48.0 were cba def recovered. Progeny with a Kinked, pink-peach phenotype carried a recombinant chromosome containing most of the rightarm euchromatin, whereas progeny with a radius incompletus phenotype carried the reciprocal recombinant product containing the entire left arm and the right-arm heterochromatin of the detachment-product chromosome. If the recombinant chromosome which included the entire left arm and the heterochromatin of the right arm were lethal over the original detachment product, thedetachment product deficiency was to the left of the Ki Pp region. If the reciprocal recombinant product failed to survive over the complete detachment, the deficiency was distal to the Ki Pp region on the right arm. FIGURE1.-Models showing the synthesis of the four possible A putative detachment product deficiency was assigned typesof compound autosomes. C(3L) chromosomes formed by to the proximal region of chromosome 3 with certainty if it sister-strand attachments are used as examples. Radiation-induced mapped to the right of the ri eg region and to the left of the breaks are indicated by arrows, 3L heterochromatin as solid blocks, Ki p’ region. and 3R heterochromatin as open blocks. The symbols a, b, and c Reattachments: To determine whether the proximal de- represent vital loci in wild-type leftheterochromatin; whiled, e, and ficiencies recovered as lethal-bearing products ofcom- fdesignate loci in wild-type right heterochromatin. (Top left) Syn- pound-3 detachment were to the left or to the right of the thesis of a C(3L) chromosome carrying no proximal deficiencies or centromere, an attempt was made to regenerate compound duplications of vital loci. (Top right) Synthesis of a C(3L) chromo- autosomes by reattaching sister chromatids of the detach- some carrying a proximal duplication (d) of 3R heterochromatin. ment pr0duct.s from several complementation classes. Virgin (Bottom left) Synthesis of a C(3L)chromosome carrying a proximal females heterozgyous for thedeficiency-bearing detachment deficiency of 3L heterochromatic region containing locus a. (Bot- chromosome over TM3 were irradiatedand mated to tom right) Synthesis of a C(3L) chromosome carrying both a prox- C(3L)VH3,st; C(3R)SH19,+ males. For each detachment imal 3R duplication and 3L deficiency. product tested, 2500 females were treated with 2500 rad and an additional 1250 females were treated with 4000 rad to know the natureof the progenitor compound autosomes. of 7-radiation. Progeny with a radius incompletus pheno- However, unlike chromosome 2 where two, recessive, visible type carried a newly formed sister-strand C(31); and those genetic markers were available to help identify some of the with a scarlet, pink-peach phenotype carried a new sister- properties of the compound autosomes, no identifying strand C(3R). Only C(31)’s were expected from detachment markers for the heterochromatic regions were available for products with a right-arm deficiency, and vice versa. chromosome 3. Therefore, it was not immediately possible Models representing the generationof polar, nonpolar to assign any of the newly generated compound-3 chromo- andcentromere-spanning heterochromatic deficiencies: somes to any of the four classes represented in Figure 1. From previous studies (HOLM1976; HILLIKER,HOLM and Any deficiency of vital loci generated on a compound au- APPELS 1982) we recognize that a compound autosome can tosome will be masked by the normal alleles on the homol- carry a duplication for proximal loci from the opposite arm, ogous arm. Itis assumed alsothat offspring with compounds a deficiency for proximal, vital loci of the same arm, both carrying a duplication are phenotypically normal. Conse- or neither. In Figure 1 we have presented four possible quently, a compound autosome can only be characterized configurations of hypothetical heterochromatic loci in com- by working backward from the patternof detachment prod- pound-31 chromosomes arising as a consequence of aniso- ucts generated. Since the frequency and type of lethal de- brachial interchanges between sister chromatids. Assuming tachments produced from a given pair of compound auto- that genetic loci do reside within the heterochromatic re- somes is a function of the interaction of the classes of both gions of chromosomes, to obtain a full understanding of the compounds within the pair, each compound autosome must distribution and relative position of these genes, it is essential be detached in several different combinations before it can Detachment Products of Compound 3 507 one carries a duplication or a deficiency. Centromere-spanning deficiencies will result from progenitor compound autosomes in which at least one is hemizygous for a vital locus in the proximal heterochromatin. The frequency of lethal detachment products generated is also partially determined by the class of the progenitor compounds. For each detachment event, there is a reciprocal detachment which would be expected to occur with an equal frequency. Each time a compound autosome carrying a deficiency for a given region is detached, one of the two reciprocal detachment products will carry the deficiency. >< Therefore, a minimum of 50% of the detachment products cb de f def from a deficiency-carrying compound will be deficient for -0- -0- -4%- the same region. If no deficiency is carried by the progenitor compounds, the proportion of detachment products deficient for a specific region cannot exceed 50%. The frequency of detachment products carrying any deficiencywill also be determined by whether or not the progenitor compounds carried duplications as well.

RESULTS Synthesis of compund-3 chromosomes: Six sister- chromatid compound-31 and three sister-chromatid compound-3R chromosomeswere recovered from approximately 1500 irradiatedfemales. Each compound

cb ef third was assigned ana-numeric code as recom- e@. mended by HOLM(1976). The three C(3R)chromosomes were designated C(3R)VMZ,pto C(3R)VM3,14; andthe six C(3L)chromosomes were designated C(3L)VMI,ri to C(31)VM6,ri. All three C(3R) chro- FIGURE2.-Models showing thegeneration of four possible mosomes and the first four C(3L) chromosomes were types of proximal deficiencies carried by detachment products. used in the detachmentexperiments. Twelve separate Irradiation-induced breaks are shown as arrows, 3L heterochro- lines were established from all possible combinations matin as solid blocks, and 3R heterochromatin as open blocks. The of these compound chromosomes. symbols a, b, and c represent loci found in wild-type left hetero- Recovery detachment products:A separate de- chromatin; while d, e, andfdesignate loci in wild-type right heter- of ochromatin. (Top left) Production of a polar deficiency (a) of the tachment experiment was undertaken for each of the left arm from progenitor compounds carrying no duplications or 12compound-3 combinations. In each experiment, deficiencies of vitalloci. (Top right)Production of anonpolar 2000 virgin females from a compound-3 strain were deficiency (b)of the left arm from progenitor compounds carrying irradiated and mated to TM3/Ly males. The offspring a duplication of locus a in the left-arm heterochromatin. (Bottom recovered were heterozygousfor thedetachment left) Production of a centromere-spanning deficiency (aand d)from progenitor compounds carrying a deficiency of left arm hetero- product over the TM3 or the Ly chromosome. The chromatin. (Bottom right) Productionof a morecomplex deficiency number of detachment products rescued in each ex- (b and e) from progenitor compounds carrying duplications and periment is shown in Table 1. Owing to the relatively deficiencies. Note the relative positions of the most proximal loci a low recovery of products in the initial cross with and d. C(3L)VM3; C(3R)VMP, experiment 6 was repeated. It will benoted that from the four detachment be properly characterized. In the present study, these objec- tives were met by undertaking separate detachment exper- experiments involving C(3R)VMP substantially fewer iments for all 12 possible combinations of three different reconstituted thirds were recovered than fromany of C(3R)chromosomes and four differentC(3L) chromosomes. the other crosses. To test whether this effect was a From each type of compound autosome, as represented result of reducedfertility in stocks carrying in Figure 1, a different pattern of detachment products C(3R)VM2, fecundity tests were carried out for each (reconstituted standard autosomes) can be obtained. Many classes of detachment products can be produced, as shown of the 12 compoundlines. Two-day-old virgin females in Figure 2, but only those with deficiencies of vitalgenetic from each line were treated with 2200 rad and mated loci can be readily identified. The types and frequencies of to males carryinga pair of compoundthirds. Six deletions generated depend on theclass of progenitor com- bottles of 25 females and 10males were examined for pounds. From compound autosomes carrying no duplica- each compoundline. The fecundity of stocks carrying tions or deficiencies of vital loci, only polar deficiencies, starting from the centromere, will be recovered (top left the C(3R)VMP chromosome proved not to differ sig- model in Figure 2). Nonpolar deficiencies will be obtained nificantly from that of the other compound stocks. only from detaching compound autosomes in which at least An alternativeexplanation was that the C(3R)VM2 508 G. E. Marchant and D. G. Holm TABLE 1 TABLE 2 Number of reconstituted third chromosomes recovered from Results of lethality tests with reconstituted third chromosomes each compound-3 detachment experiment' from each detachment experiment

No. of No. Expt. detachments Expt. of No. of Percent No.C(3R) C(3L) recovered No. C(3R)C(3L) lethals nonlethals lethals" 1 VM2 VM 1 218 1 VM2 VMI 40 79 33 2 VM4 VM2 108 2 VM4 VM2 53 31 63 3 VM3 VMl 166 3 VM3 VMI 66 38 63 4 VMI VM 1 20 1 4 VMI VMl 38 116 25 5 VMl VM2 112 5 VMI VM2 42 47 47 6A VM3 VM2 80 6 VM3 VM2 42 16 72 6B VM3 VM2 98 7 VM4 VM3 62 31 67 7 VM4 VM3 178 8 23 VM4 109VMI 33 8 VM4 VMI 144 9 VM2 32 VM2 39 55 9 VM2 VM2 93 10 VM3 VM3 6862 29 10 VM3 VM3 218 11 VMl VM3 5050 51 11 VM 1 VM3 163 12 VM2 39 VM3 41 51 12 VM2 VM3 238 The percent ofall detachment products tested that carried ' In each experiment, 2000 compound-3 females were treated recessive lethals. with 2200 rad of y-radiation and mated with TM3/Ly males. METHODS. First, a separate complementation map was chromosome carried a massive deficiency of proximal produced for experiments 1,2, 3,4, and 10 by testing heterochromatin, and thereby offered a smaller target the lethal detachment products of each experiment in to y-rays. Subsequent findings reported below failed all inter se combinations. Testing between complemen- to substantiate this proposal. Nevertheless, the possi- tation groups from the different experiments pro- bility that some intrinsic property of compound au- duced an accumulated complementation map for the tosomes may influence their detachment frequencies five experiments. Finally, an accumulated comple- deserves further attention. mentation map for all twelve experiments was pro- Detachment products recovered in malesor females duced by testing all the lethal detachment products over TM? and in males over LJ were maintained in from the remaining seven experiments against tester stock balanced over TM3. Each detachment product chromosomes from each complementation group in was assigned a code number which identified the the five-experiment complementation map. experiment, and hence the compounds from which Figure 3 represents the accumulated complemen- they were derived, and the detachment product. For tation map of the lethal detachment products from all example, 2-10 is the tenth detachment product re- 12 experiments. Each complementation group is rep covered from experiment 2. resented by a solid bar; complementation groups Lethality tests: The results of lethality tests with shown to overlap fail to complement. Above each bar the detachment products tested from each experiment the complementation group is designated by the code appear in Table 2. No apparent semilethality was number of a representative detachment product. The detected and no sterility was observed. It can be seen number under thebar represents the number of chro- from the data that the percentage of lethal-carrying mosomes that fall into that particular complementa- detachment products varied considerablyfrom exper- tion group. iment to experiment. In three experiments, less than As shown in Figure 3, 18 complementation groups 35% of the detachment products carried lethals; while were identified with as many as 146 and as few as 1 in five others, greater than 60% of the chromosomes detachment product per group. This initial comple- were homozygous lethal. The other four experiments mentation map suggested that at least eight, distinct, were within 5% of 50% lethals. These differences in vital loci were uncovered by the putative deficiencies lethal frequencies likely reflect different classesof on the detachment chromosomes. Small complement- progenitor compounds. At least 25 lethal detachment ingdeficiencies specific for sevenof these regions products from each experiment were maintained in were indicated, and the presence of an eighth region stock and usedin the complementation analysis. A between the 3-30 and 3-9 groups was implied by the total of approximately 150 nonlethal detachment complementation pattern. The two complementation products were also saved. groups shown as two solid blocks joined by a dotted Complementation analysis: A total of 443 lethal linewere interpreted as detachment products that detachment products from the 12 separate experi- carried double deficiencies, one on each side of the ments were tested for complementation following the centromere. Such centromere-spanning double defi- several-step procedure described in MATERIALS AND ciencies could only be generated by progenitor com- Detachment Products of Compound 3 509

68 -29 1

,-,"""""""""""""~-~ 10- 26 2

10-42- """--,-, -1

10-39 58

I- 166 4

2- 66 81

10-68 3

3- 126 4-184 2 3

4- 134 9-56 74 2

3- 144 6-61 8A-80 3-30 3-9 10- 65 1-168 "" 2 4 2 1 "-7 146 5 FIGURE3.-Accumulated complementation map of putative deficiencies carried by detachment products from all 12 detachment experiments. Each bar represents a different complementation group. Bars that do not overlap represent complementing groups, and vice versa. The number above each bar designates the detachment product used to represent the complementation group. The number below the bar indicates the number of detachment products assigned to the complementation group. Dotted lines represent discontinuous deficiencies. See text for further details. pound chromosomes carrying a deficiency. The 68% third hits responsible for generating lethals elsewhere frequency oflethals among experiment 10 detach- on the chromosome. ments is consistent with this. It should be noted that, One additional detachment product included in the to this point, the arrangementof the complementation complementation analysis, but not shown on thecom- map drawn in Figure 3 is to some degree arbitrary. plementation map, was lethal 22-64. This detachment Other variations of this map are also consistent with chromosome was semilethal over all deficiencies of the results. However, theseambiguities willbe re- the region defined by lethal 3-30, and surviving het- solved below. erozygotes had slightly outspread wings with only a In addition to thedetachment chromosomes shown partial posterior cross-vein. The 22-64 chromosome on the complementation map, 44 chromosomes be- fully complemented detachment products from all haved as random hits, that is, mutational events other other complementation groups. Unfortunately, this than those involvedin generating the detachment. stock was lost, making further tests impossible. These chromosomes complemented all others against Vials containing detachment products of the 3-9 which they were tested. Twenty of the putative ran- complementation classalso exhibited a phenotype. dom hit lethals from different experiments were se- Rare homozygous adults eclosed, but died within 48 lected and complementation tested in all inter se com- hr of eclosion. Surviving adults had a phenotype re- binations. All crosses resulted in complementation, sembling rotund, with droopy wings and reduced sex providing further evidence that during the detach- combs in males.Nonetheless, all detachment products ment event these chromosomesreceived random, ofthis class fully complemented rotund, whichhas 510 G. E. Marchant and D. G. Holm been mapped to band 84D on the right arm of chro- 144 are all from experiment 3. Two of these lethals, mosome 3 (DUNCANand KAUFMAN1975). The phe- from the 3-126 complementation group, also failed notype expressed by homozygotes of the 3-9 comple- to complement many other deficiencies in the proxi- mentation group was designated rotund-like. mal region. Recombinational analysis of detachment- A number of nonlethal detachment products were product 3-126 disclosed two separable lethal sites on also testedfor complementation with some of the this chromosome, one in the proximal region and one lethal complementationgroups. If theheterochro- in the right euchromatin that failed to complement matin of chromosome 3 contains tandemly repeated 3-144. Therefore, it would appear that a spontaneous genes, it is possible that partial deletions would be lethal arose inthe right arm euchromatinof a subpop- homozygous viable. However, partial deletions may ulation of the C(3R)chromosome used in experiment be lethal over more severe deficiencies. To test this 3. Ofthe four detachment products carrying this possibility, approximately 150 nonlethal detachment lethal, two also carrieda proximal deficiency that products were each crossed to lethals 3-126, 1-166 arose during the detachment event. and 10-65, which collectively span the accumulated Lethals on fourof the 44 detachmentchromosomes complementation map. However, all the crosses re- initially classified as random hits were also located sulted in full complementation, andthe non-lethal through recombinational analysis. Three of the four detachment products were discarded.As well, to keep lethals mapped outside the proximal region, but the stocks manageable,a maximum of tenlethal lines fourth, designated 10-33, mapped to the proximal continued to be maintained for each complementation region. This chromosome was tested against all re- group. maining lethal detachment products in complemen- Recombinationmapping: The proximal position tationgroups that possibly extendedfurther than of the putative heterochromatindeficiencies was con- what had been revealedby the complementation map. firmed using the recombinationmapping protocol Three of the ten strains from the 10-39 complemen- described in the MATERIALS AND METHODS section. tation group andfive of the ten from the4-134 group Exchangeevents between tightly linkedproximal failed to complement 10-33. Therefore, 10-33 de- markers on either theleft or right armwere recovered fines a new complementation group that was previ- from females heterozygous for a detachment product ously undetected because the testerchromosomes and a marker chromosome. For most reconstituted used to represent the 10-39 and 4-134 complemen- thirds, the lethal deficiency always remained with the tation groups did not include 10-33. Consequently, recombinantproduct containing the centromeric both the 10-39 and the 4-134 groups must be rede- fragment of the detachment chromosome. This was fined as containing at least two subgroups, one that the case for detachment products, 10-65, 10-42, 4- includes the 10-33 region and one that does not. In 134, 10-39, 6B-29, 3-9, 6-61, and 4-75. These de- view of this finding, mapping studieswere conducted ficiencies, which include most of the complementation on an additional ten chromosomes whose lethals had groups, are therefore definitely located in the proxi- been classified as random hits. However, the results mal region of chromosome 3. were in agreement with the original classification as The lethals of detachment products 1-168 and 3- no additionalcomplementation groups were dis- 144 (Figure 3), however, mapped to the right arm closed. euchromatin. The complementation grouprepre- Reattachment of detachment products: Deficien- sented by putative deficiency 1-168 consists of five cies associated with detachmentproducts were as- lethal detachments, all recovered in experiment1. signed either to the left or to the right arm hetero- The lethals in this group complement all other com- chromatin from the results of generating compound plementation groups. It should be noted that all 12 autosomesfrom thedetachment chromosomes. Fe- compound strains used in this study had been main- males heterozygous for TM3 and one of the detach- tained in stock for close to a year prior to detachment. ment chromosomes, selected from the various com- It seems likely that a spontaneousrecessive lethal arose plementation groups, were irradiated and mated to in theright-arm euchromatin and spread within a males carrying compound thirds. Foreach deficiency- subpopulation of the experiment 1 compound stock. bearing chromosome, 2500females were treated with Consequently, approximately 50% of the detachment 2500 rad and anadditional 1250 females were treated products generated from females bearing the lethal with 4000 rad of y-radiation. In addition to those mutation would also carry the lethal. This explanation offspring arising from nondisjunctional events, indi- is consistent with the finding that spontaneous reces- viduals were recoveredwith a newly synthesized sister- sive lethals do accumulate on compound chromsomes strand compound-3 chromosome. Since sister-strand (PARKER 1954). attachments are homozygous for one of the chromo- A similar explanation is likely for lethal 3-144. The somal arms of the lethal-bearing detachment chro- three otherchromosomes that do notcomplement 3- mosome, only C(3L)’sshould be rescued from detach- Detachment Products of Compound 3 51 1 TABLE 3 arrangements. Asimilar finding had been observed in Sister-strand compound thirds recovered from the radiation- the original detachment experiment, where one par- induced reattachmentof selected detachment products ticularcompound chromosome exhibited a significantly reduced propensity to be detached. These re- Sister-strand compound sults suggest that third chromosomes previously in- recovered” Detachment volvedin attachment-detachmentevents with product WLL) C(3W heterochromaticbreakpoints differ widelyin their 3-9 0 5 competency to be involved in further rearrangements. 3-30 0 6 A control reattachment experiment was conducted 1-16 0 2 by reattaching the isogenic ri pf’ chromosome while 1-166 0 4 2-66 0 2 balanced over TM3. Since the ri pf’ chromosome does 10-26 0 0 not carry any recessive lethals, it should produce new 10-42 0 0 sister-strand attachments of both the left and right 10-65’ 5 1 arms. As predicted, both C(3L)’s and C(3R)’s were 10-65b 4 0 recovered in approximately equal numbers. 1-168 21 0 ri pp’ 6 8 Further analysis of compound and detachment chromosomes: Figure 4 contains the revised comple- A total of 3750 heterozygous females were treated for each reattachment test. See text for details. mentation map that includes the results of the recom- This experiment was repeated owing to the unexpected recov- binationmapping andreattachment experiments. ery of a C(3R)chromosome in the first reattachment test. This map suggests the presence of six proximal loci As a controlsister-chromatid attachments were generated from the isogenic ri Pp chromosome balanced over TM3. on the left arm and one on the right. The right arm locus is designated 1(3)Rhl, and the six lethal loci on ment products carrying adeficiency of the right arm, the left arm are numbered Z(3)Lhl to Z(3)Lh6, where and vice versa. The results of the reattachment exper- the h designates heterochromatin. Although thecom- iments are summarized in Table 3. Only new C(3R)’s plementation map is shown with Z(3)Lhl proximal and were generated from tester chromosomes represent- 1(3)Lh6 distal on the left arm, which is the simplest ing the 3-9, 3-30, 1-16, 1-166 and 2-66 complemen- interpretation, the results to this point cannot distin- tationgroups. Therefore, thesecomplementation guish between this possibility and the opposite orien- groups and others that fail to complement them are tation. However, the orientation of the deficiencies presumed to represent deficiencies of the left arm onthe left arm can bedetermined by genetically heterochromatin. As expected, the 10-26 and 10-42 characterizing the originalcompounds used in the chromosomes failed to produce any new sister-strand detachment experiments, since deficiencies or dupli- attachments of the left or right arm, confirming that cations carried by the compounds will be polar (ie., thesedetachment chromosomes carry double defi- they will include the most proximal gene and extend ciencies. distally). Individual complementation maps for each The 10-65 chromosome reattachment experiment of thetwelve detachment experiments were produced was repeated because, in the first experiment, five from the overall map, and then analyzed for clues new sister-strand C(3L)’s and one new C(3R) were about thegenetic makeupof the compounds that were recovered. The recovery of compounds from both progenitors. arms would have suggested that the 10-65 chromo- If an ancestral compound carried adeficiency, over some carried a lethal on neither arm, which is clearly 50% of all detachment-products from that compound notthe case. To resolve this anomaly, the 10-65 would carry a deficiency for the same specific region. reattachment experiment was repeated, and this time Analysis of the data revealed that over 50% of the only new C(3L)’swere recovered. It is evident that detachment chromosomes producedin all four exper- the 10-65 chromosome does carry lethal a on the right iments involving C(3R)VM3 carried deficiencies of arm. The single C(3R) recovered in the first experi- Z(3)Rhl. Therefore, C(3R)VM3 was heterozygous for ment remains unexplained. a deficiency of l(3)Rhl.Similarly, two of three exper- Reattachment results with the 1-168 chromosome iments using C(3L)VM3 produced over 50% lethals are also peculiar. As explained above, this chromo- for a specific region on the left arm, indicating that somecarries a lethal outside the proximalregion C(3L)VM3carried adeficiency of left arm heterochro- somewhere in the right euchromatin and this is con- matin. The thirdexperiment involving this com- firmed by the recovery of only C(3L) chromosomes. pound, which produced fewer than 50% lethals for However, the relatively large recovery of reattach- any region, must have involved a C(3R) carrying a ments is difficult to explain. It appears that this de- duplication that compensated for the deficiency car- tachment chromosome differs significantly from the ried on C(3L)VM3. The centromere-spanning defi- others tested in its ability to undergofurther re- ciencies produced in experiment 10, where C(?R)VM3 512 G. E. Marchant and D. G. Holm 60- 29 ...... t-b 1

...... -70-42 1

9-52 (17)

10-39

1 - 166 4

2-66

10-68 3

1- 16 4- 184 (37) 3

4-134 9-56 2

10-33 6-67 8A-80 3 -30 3-9 10-65 "" 4 - 1 1 - 2 7 146

I I I I I I m I w I (3)1h6 I(3)lh5 I (3jlh4 I(3)lhZ1(3)lh3 I (3Ilh7 Ic3)Rhl

FIGURE4.-Revised complementation map incorporating results of recombination mapping and reattachment experiments. The number above each bar designates the detachment product used to represent each complementation group; and the number below the bar indicates the number of detachment products assigned to each group. The number of detachment products in the 4-134, 1-16, 10-39, and 9-52 complementation groups is shown in brackets because they are estimates. Estimates were necessary because only some of the detachment products in these groups were available for complementation tests with the previously undetected 10-33 group. was paired with C(jL)VM3, were diagnostic of defi- without first knowing the proximal-distal orientation ciency-carrying compounds and confirmed that at of the deficiencies. To solve both unknowns simulta- least one of the two compounds carried a deficiency. neously, a set of possible models of the progenitor Moreover, from experiment 6, the centromere-span- compound chromosomes was constructed and ana- ning deficiency places the break in (C3L)VMj to the lyzed to determine if it could account for all the region separating 1(3)Lh5 and 1(3)Lh6. Deficiencies detachment-product classes generated in the 12 ex- were not associated with any ofthe remaining progen- periments. The only configuration that could explain itor compound autosomes. all 443 detachment products had the orientation of Any detachment experiment that does not involve the left arm as shown in Figure 4 and the progenitor a deficiency-carrying compound, but which results in compounds described in Table 4. Using this set of nonpolar detachment deficiencies must involvea pro- progenitor compounds and orientation of the left arm, genitor compound carrying a duplication. However, possible breakpoints were identified for every detach- it is not possible to identify all nonpolar deficiencies ment chromosome. Detachment ProductsDetachment of Compound 3 513

TABLE 4 TABLE 5 Duplications anddeficiencies carried by progenitor compound- Rescue of homozygous deficient sister-strand compound-3 3 chromosomes as predicted from theanalysis of detachment chromosomes by progenitor compounds' products Homozygous Progenitor Duplication Deficiency deficient compound-3's C(3L)VMI None None Deficiency rescued Progenitor chromosome Predicted C(3L)VM2 None None compound tested duplications C(3R) C(3L) C(3L)VM3 None 1(3)LhI-5 C(3L)VM4 None None C(3L)VMl 10-65 None 0 C(3R)VMl 1(3)Lh1-3C(3R)VMl None C(3L)VM2 10-65 None 0 C(3R)VM2 None None C(3L)VM3 10-65 None 0 C(3R)VM3 1(3)LhI-6 l(3)Rhl1(3)LhI-6 C(3R)VM3 C(3L)VM4 10-65 None 0 C(3R)VMl 3-9 I(3)Lhl-3 5 C(3R)VMP 3-9 None 0 Two ambiguities remained for thecompound chro- C(3R)VM3 3-9 l(3)Lhl-6 6 4. mosome configurations described in Table First, In each of the seven experiments, 8000 radiation-treated fe- the duplication carried by C(3R)VM3could extend to males were used. 1(3)Lh5 or 1(3)Lh6. Both possibilities were consistent with the data, andcould not be distinguished further. C(3R)VMl and C(3R)VM3 were the only progenitor The second ambiguity arose from the uncertainty of compounds carrying duplications of vital loci on prox- whether any of the four C(3L)chromosomes carried imal 3L and supported the polarity of the map pre- duplications. With only one complementation group sented in Figure 4. on the right arm, it was not possible to identify non- Distribution of putative heterochromaticloci: The polar deficiencies that would be diagnostic for a du- breakpoints involved inthe formation of the complete plication carried on C(3L). This ambiguity was re- set of detachment products are not evenly distributed solved by a reattachment experiment which tested the along the complementation map. Some regions of the capability of progenitor compounds to rescue homo- map have a high frequency of breakpoints whereas zygous deficient sister-strand attachments. As de- breakpoints in other regions are rare. If the distance scribed above, no C(3R)sister-strand attachment will between two genes is assumed to be proportional to be recovered from a detachment product carrying a the number of breakpoints that occur in that region, deficiency on the right arm, andvice versa. However, it is possible to construct a distribution map of the when females carrying a detachment product with a genes in the thirdchromosome heterochromatin (Fig- deficiency on the right arm balanced over TM3 are ure 5). irradiated and mated to males carrying a C(3L) with One difficulty in producing such a map was that a duplication of the right arm, the duplication should many experiments involving a progenitor compound be able to rescue homozygous deficient sister-strand carrying a duplication or deficiency provided two or C(3R)'s. more possible sets of breakpoints for generating the Each of the seven progenitor compounds used in same detachment chromosome. Therefore, when cal- the detachment experiments was tested for the pres- culating the map distances for either the left or right ence of duplications by this method. The results of arm, experiments that involved compounds carrying the reattachment experiments are shown in Table 5. a deficiency or duplication for the arm being exam- Each of the four C(3L) chromosomes was tested by ined were excluded from the analysis. As a result, the mating 8000 irradiated females, heterozygous for the only experiments used in the calculations were those right arm deficiency 10-65, to males carrying the where the breakpoints could be assigned to a specific C(3L)VM chromosome being tested. Offspring were region with complete certainty. screened forthe presence ofnewly synthesized For the right arm, fourexperiments involved com- C(3R)'s.Since no sister-strand C(3R)'s were recovered, pounds with a defikiency of the right arm and thus thefour progenitor C(3L) chromosomesmust not had to be excluded from the analysis. In the remaining carry duplications of the region defined by the 10-65 eight experiments, only 22 of a total of 649 detach- deficiency. ment products were deficient for the gene(s) defined The three C(3R)VM chromosomes were tested in a by the 10-65 deficiency. Ifa breakpoint occurs on the similar manner. Females heterozygous for theleft arm right arm proximal to the 10-65 region, none of the deficiency 3-9 (Figure 4) were treated with 4000 rads resulting detachment-products will carry a deficiency and mated to males carrying a progenitor C(3R)VM for this region. If, on the other hand, thebreakpoint chromosome. Newly synthesized sister-strand C(3L)'s occurs distal to the 10-65 region, then only one-half were rescued by C(3R)VMI and C(3R)VM3, but not of all detachment products will be deficient for this by C(3R)VM2. These results confirmed that region. That is,assuming an equal probabilityof 514 G. E. Marchant and D. G. Holm

No. of detachments) X 100% = 2 (22)/649 x 100% = 7%. .23 Therefore, on thebasis of breakpoint frequencies, the 10-65 regionis located at a position 7% of the distance .Ol fromthe euchromatic-heterochromatic junctionto the centromere. Similar calculations were made for each locus on the left arm. Only data from experiments 2, 5 and 9 were used, since these were the only experiments not .67 involving compounds carrying duplications or deficiencies of the left arm. The relative positions of l(3)LhI,1(3)Lh2, 1(3)Lh3, 1(3)Lh4, and l(3)Lh5 were 91%, 87%, 85%, 24% and 23% respectively of the distance from the left euchromatic-heterochromatic junctionto the centromere. The map position of .02 .04 l(3)Lh6 was not calculated owing to theextremely low .09 number of detachment products tested for a defi- -*- ciency of this site. However, a very rough estimate would place it half-way between l(3)Lh5 and the left euchromatic-heterochromatic junction. The map distances shown in Figure 5 are proportional to the numberof radiation breakpoints induced in each region.However, these map distances are proportional to the actual physical distances between loci if and only if radiation breakpoints are randomly .93 distributed within the heterochromatic block. Lethal phases: The lethal phases for several of the detachment-product deficiencies were determined using egg count experiments. Females carrying a deficiency over TM3 were mated to sibling males and then allowed to lay eggs forthree successive two-hour periods on petri dishes containing fresh medium.The final petri dish in each series was examined daily. All deficiencies testedhad larval lethal phases during I(3)Rhl either the second or thirdinstar stages. -07 -+ E-H JUNCTION FIGURE5.-Distribution of genes in the proximal heterochro- DISCUSSION matin of chromosome 3. Relative map distances are based on the number of radiation breakpoints induced in each region. See text Inthe experiments described above,a series of for details. compound-autosomeattachments, detachments and reattachments were generated and used to dissect the recovering either the centric or acentric fragment, heterochromatin of chromosome 3 of D.melanogaster. one of the two detachmentproducts will carrya The analysis of approximately 400 proximal deficien- duplication of the 10-65 region, thereciprocal detach- cies carried by detachmentproducts, revealed the ment-product will carry a deficiency. Therefore, the presence of at least seven vital genetic lociin the actual frequency of breakpoints that occur distal to a proximal region of chromosome 3. Attempts to reat- particular locus is twice the frequency of deficiencies tach chromosomes carryingdeficiencies of these genes recovered for that locus. If it is assumed also that established that six of the genes were on the left arm detachment-product breakpoints occuronly in heter- and that one was on the right. Thisstudy has further ochromatin and are randomly distributed within the demonstrated the usefulness of compound-autosome heterochromatic block, then the distance of the 10-65 attachment and detachment procedures foranalyzing region relative to the centromere from the euchro- the proximal regions of Drosophila autosomes by pro- matic-heterochromatic junction is calculated as fol- viding a genetic map of what has been until now the lows: largest uncharted region of the D. melanogaster genome. 2 (No. of detachments deficient forIU-65/total Several modifications of the compound attachment Detachment Products of Compound 3 515 and detachment procedures were used in the course cytological and genetic analysisof numerous com- of this study. First, reattachment of deficiency-carry- pound-2 chromosomes, even those carrying duplica- ing detachment products identified the positionof tions for the rl or It genes, failed to disclose duplica- deficiencies relative to the centromere. Second, the tions for euchromatic loci (HOLM1976). The results ability of compound autosomes to rescue newly syn- of the present study are also consistent with a heter- thesized, homozygous-deficient, sister-strand attach- ochromatic location for vital genes uncovered by de- ments was used to test for the presence of duplications tachment-product deficiencies. Examinations of poly- on compound chromosomes. Finally,the use of many tene chromosomes from larvae carrying detached different combinations of compound chromosomes in thirds revealed no obvious duplications or deficiencies detachment experiments permitted the genetic char- of the proximal euchromatic bands, and observations acterization of the progenitor compounds and, there- of metaphasepreparations of three detachment prod- fore, a detailed and useful breakpoint analysis of all ucts (MARCHANTand HOLM1988) verified that ge- the resulting detachment products. netic deletions of the more proximal loci correspond Large-scale production of detachment products to cytologicaldeficiencies restricted tothe hetero- generated from various combinations of compound chromatic blocks. autosomes, heterozygous for deficiencies and (or)du- The genetic map presented in Figure 5for the plications ofthe proximal heterochromatin, permitted relative position of vital loci in the proximal hetero- the recovery of five complementing deficiencies on chromatin of chromosome 3 portrays a very uneven the left arm, and, through overlapping deficiencies, distribution of breakpoints along the complementa- the recognition of six complementing regions. In the tionmap. If we assume thatthe physical distance absence of preexisting duplications or deficiencies on between any two genesis proportional to the number the compounds, knownvisible genetic markers or of radiation induced breakpoints that occur in that semilethality, asfound by HILLIKERand HOLM(1 975) region, and that all regions of the heterochromatin for chromosome 2 heterochromatin, all deficiencies are equallysusceptible to radiation-induced inter- would be polar, and consequently overlapping. For change, then, as shown in Figure 5, the genes to the the segments that lie distal to region 6 on the left arm left of the centromere occur in clusters throughout and region 1 on the right, no further resolution is the heterochromatin. The single identifiable locus to possible by the detachment technique without corre- the right of the centromereis close to theheterochro- sponding duplications or preexisting deficiencies. The matic-euchromatic boundary. absence of resolution within the right heterochroma- However, from other studies, there is some limited tin suggeststhat possibly onlyone genetic locus resides experimental evidence that radiation-induced break- in this region and the low frequency with which it is points are not evenly distributed within heterochro- uncovered through detachments implies that it occu- matin. Visual examinations of a series of radiation- pies a distal site in the right heterochromatin. Genes induced rearrangements with heterochromatic break- proximal to the mostproximal complementation points detected a preponderance of interchanges at group on either side, but mainly to the right, of the or near the euchromatic-heterochromatic junction centromere, may not have been detected if they were (GATTI,TANZARELLA and OLIVEIRI 1974; SCHUBERT tandem repeats. However, after testing 150 nonlethal and RIECER 1976). KENNISON(1 98 1) also observed a detachments against the largest deficiencies that col- striking nonrandom distribution of translocation lectively spanthe complementation map, we found no breakpoints on the heterochromatic Y chromosome, evidence to support the notion of tandemly repeated with most if not all breakpoints within nonfluorescent vital loci within the deleted segments of heterochro- segments, or near junctions between differentially matin. staining regions. If radiation breakpoints in hetero- There is convincing evidence that the genes uncov- chromatin are nonrandomly distributed and possibly ered by detachment-product deficiencieslie within clustered near theeuchromatic-heterochromatic junc- heterochromatin. HILLIKERand HOLM(1975) found tion, then the radiation breakpoint map in Figure 5 is deficiencies on chromosome 2 to be genetically prox- skewed toward the centromere, and thereal positions imal to previouslyisolated deletions that extended of the genes are more distal than shown. Extensive from the euchromatin into the distal heterochroma- cytologicalanalysis of heterochromatic deficiencies tin. Similarly, the deficiencies of the third chromo- generated in standard chromosomes will be required some recovered by BALDWINand SUZUKI(1 97 1) com- to disclose the exact location of these genetic loci. plemented all known proximal euchromatic markers In connection with the generation of rearrange- on the third chromosome. Cytological observationsof ments and the distribution of breakpoints in the het- polytene chromosomes carrying detachment-product erochromatin, two other findings merit attention. The deficiencies of chromosome 2 did not detect removal first is that attached and detached chromosomes can of any euchromatin (HILLIKER andHOLM 1975), and differ widely in their propensity to undergo further 516 Marchant G. E. and D. G. Holm rearrangements. In the detachmentof compound au- BROWN,S.W.', 1966 Heterochromatin. Science 151: 417-425. tosomes, all experiments involving C(3R)VMB consist- BRUTLAG,D., R. APPELS,E. S. DENNISand W. J. PEACOCK, 1977 Highly repeated DNAin Drosophilamelanogaster. J. ently generated significantly fewer detachment prod- Mol. Bioi. 12: 31-47. ucts than experiments involving the other compound COHEN,E. H., and S. C. BOWMAN,1979 Detection and location autosomes. Similarly, the 3-126 detachment chromo- of three simple sequence DNAs inpolytene chromosomes from some was several times more likely to undergo reat- virilis group species of Drosophila. Chromosoma 73: 327-355. tachment than the other detachment products tested. DUNCAN,1. W., and T. C. KAUFMAN,1975 Cytogenetic analysis of chromosome 3 in Drosophila melanogaster: mapping of the The second observation of possible significance is proximal portion of the right arm. Genetics 80 733-752. that detachment products carrying a largedeficiency DURICA,D. S., and H. M.KRIDER, 1978 Studies on the ribosomal tend to carry a compensating duplication. Three de- RNA cistrons in interspecific Drosophila hybrids. 11. Hetero- tachment products fromthis study that were analyzed chromatic regions mediating nucleolar dominance. Genetics by S. PIMPINELLI(personal communication), tobe 89 37-64. GALL,J. G., and D. D. ATHERTON,1974 Satellite DNA sequences discussed in more detail in the next paper(MARCHANT in D. virilis.J. Mol. Biol. 85: 633-664. and HOLM1988), contained relatively large deficien- GANETSKY,B., 1977 On the components of Segregation Distor- cies compensated by large duplications. As a result, tion in Drosophila melanogaster. Genetics 86 321-355. there was an approximately normal-sized heterochro- GATTI,M., and S. PIMPINELLI,1983 Cytologicaland geneticanaly- matic block on both sides of the centromere of each sis of the Y chromosome of Drosophila melanogaster. Chromo- soma 88: 349-373. reconstituted, standard, third chromosome.HILLIKER GATTI,M., C. TANZARELLAand G. OLIVIERI,1974 Analysisof and HOLM(1975) observed a similar phenomenon the chromosome aberrations induced by X-rays in somatic cells with second chromosome heterochromatic deletions. of Drosophila melanogaster. Genetics 77: 70 1-7 19. Althoughtheir reconstituted second chromosomes COLDSTEIN,L. S. B.,R.W. HARDYand D. L. LINDSLEY, carried genetically large deficiencies, the mitotic chro- 1982 Structural genes on the Y chromosome of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 79 7405-7409. mosomes appeared normal, suggesting that the dele- HARDY, W.,R. K. T. TOKUYASUand L.D. LINDSLEY, tions were compensated by accompanying duplica- 198 1 Analysis of spermatogenesis in Drosophila melanogaster tions. Additionally, from the genetic results of this bearing deletions for Y-chromosome fertility genes. Chromo- study, the deficiency of region 1 in the right hetero- soma 83: 593-617. chromatin of C(3R)VM3was compensated by a dupli- HARDY,R. W., D.L. LINDSLEY,K. L. LIVAK,B. LEWIS,A. L. SILVERSTEN,G. L. JOSLYN,J. EDWARDSand S. BONACCORSI, cation of regions 1 to 6 from theleft heterochromatic 1984 Cytogenetic analysis of a segment of the Y chromosome block. It seems unlikely that chromosomes with little of Drosophila melanogaster. Genetics 107: 591 -610. or no heterochromatin on one arm cannotbe rescued, HAZELRIGG,T., P. FORNILIand T. C. KAUFMAN,1982 A cyto- as Df(2R)MS2-10 (MORGAN, SCHULTZand CURRY genetic analysis of X-ray induced male steriles on the Y-chro- 1940), even though not a detachment product, carries mosome ofDrosophilamelanogaster. Chromosoma 87: 535-559. HILLIKER,A. J., 1976 Genetic analysis of the centromeric heter- a cytologically visible deficiency of most, if not all, ochromatin of chromosome 2 of Drosophila melanogaster: defi- rightarm heterochromatin (HILLIKERand HOLM ciency mapping of EMS-induced lethal complementation 1975). Therefore, the apparent conservation of het- groups. Genetics 83: 765-782. erochromatic symmetry flanking the centromere sug- HILLIKER,A. J., and R.APPEIS, 1982 Pleiotropic effects associ- gests something intrinsic about the process of attach- ated with the deletion of heterochromatin surrounding rDNA on the X chromosome of Drosophila. Chromosoma 86 469- ing and detaching compoundautosomes. 490. HILLIKER,A. J., and D. G. HOLM,,1975 Genetic analysis of the We gratefully acknowledge KAREN MORIN for preparing the proximal region of chromosome 2 of Drosophila melanogaster. figures in this paper. G.E.M. was the recipient of a Natural Sciences I. Detachment products of compound autosomes. 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