Copyright 0 1988 by the Society of America

Genetic Analysis of Chromosomal Region 67A-D of Drosophila melanogaster

Brenda G. Leicht' and J. JosC Bonner2 Department of Biology, Indiana UniversiCy, Bloomington, Indiana 47405 Manuscript received December 17, 1987 Accepted March 3, 1988

ABSTRACT Inan effort to (1) characterize the 67 interval of chromosome 3 of Drosophila melanogaster genetically and(2) isolate of the 67B1 small heat shock protein (hsp) gene cluster specifically, we undertook a mutational analysis of the 67A-D subinterval. Using a deficiency of the 67A2 to 67D11-13 region, Df(3L)ACI, we screened 8700 diepoxybutane-treated chromosomes and 7800 ethyl methanesulfonate-treated chromosomes for visible and lethal mutations throughout this interval and recovered 74 independent recessive lethal mutations, but no visible mutations. One of the lethal mutations, d29A6, was identified as an overlapping deficiency extending from 66F3 to 67B1. An additional 6000 diepoxybutane-treated chromosomes were screened for lethality over d29A6, yielding anotherfour lethal mutations within the 67A2-Bl subinterval. These 78 lethal mutations, along with two others isolated in other laboratories, define 23 essential loci-6 within the 67A2-Bl subinterval and 17 within the 67A2 to Dl 1-13 subinterval. Many of these loci appear to be required for imaginal development only, exhibiting late larval to pharate adult lethal phases. Examination of the 67A2-Bl lethal complementation groupsfor (1) earlier onset of lethality following a heat shock, (2) missing or altered small hsps on two-dimensional protein gels, and (3) restoration of viability by transformed wild-type copies of the small hsp genes indicates that none of these mutations affect the small hsps. On the basis of this analysis and the known homology of the genes, we conclude that the small hsps are functionally equivalent.

HE availability of sophisticated genetic, cytolog- (SHEARNet al. 1971; SHEARN,HERSPERGER, and HER- T ical, and, more recently, molecular techniques SPERGER 1978). The recessive wing curooid has allowed characterization of a considerable portion [cur] had also been mapped to approximately 67C of the Drosophila melanogaster genome (for example; (30.0 map units), but subsequent investigation has seeGAUSZ et al. 1981, 1986;HILLIKER et al. 1980; called this map position into question (LEICHT1987). HOCHMAN1976; JUDD, SHEN, and KAUFMAN 1972; In an effort to characterize the 67 region of D. KAUFMAN,LEWIS, and WAKIMOTO1980; KOTARSKI, melanogaster further, we have carried out screens for PICKERT,and MACINTYRE 1983;LEFEVRE 1981; ROB- diexpoxybutane (DEB)- and ethyl methanesulfonate ERTS et al. 1985; WOODRUFFand ASHBURNER 1979). (EMS)-induced recessive visible and lethal mutations Nonetheless, many regions of the Drosophila genome throughout the 67A2 to 67D11-13 subinterval. As a still remain largely uncharacterized, both genetically second goal of this analysis, we have attempted and molecularly. Among the regions for which little specifically to isolate mutations of the small heat characterization has been reported is the 67 interval shock protein (hsp) genes. These four genes, along of chromosome 3. Although this region contains 56 with three closely related genes (gene 1, 2, and 3), chromosome bands (BRIDGES 1941),very few known are tightly clustered in a 15-kb region at polytene genetic loci have beenmapped to this region (see chromosome band 67B1 (CORCESet al. 1980; CRAIG Figure 1). Amongthe few genes reported to map and MCCARTHY1980; VOELLMYet al. 1981;AYME within the 67 intervalare thesmall heat shock protein and TISSI~RES1985). In addition to their induction genes at 67B (hp28,hsp26, hp23, and hp22; PETER- by heat and other environmental stresses (reviewed SON, MOLLERand MITCHELL1979; CORCESet al. 1980; in ASHBURNER and BONNER1979), these seven genes CRAIG andMCCARTHY 1980; VOELLMYet al. 1981), a are developmentally regulated (SIROTKIN and DAV- maternallyexpressed a-tubulin gene at 67C (KAL- IDSON 1982; IRELANDet al. 1982; CHENEY and SHEARN FAYAN and WENSINK198 1; K. MATTHEWS,personal 1983; ZIMMERMAN,PETRI and MESELSON 1983; communication), Minute(3)i [M(3)i]at 67C (Moscoso MASON,HALL and GAUSZ1984; AYMEand TISSI~RES DELPRADO and RIPOLL1983), andthe vital locus 1985). While it is thought that the small heat shock 1(3)67Fa (also known as polycombeotic [pco]) at67F proteinsperform some vital function duringheat shock, and possibly during normal development as ' Presentaddress: Department of Molecular Genetics, 484 W. 12th Avenue, The Ohio State University, Columbus, Ohio 43210. well, the specific function(s) of theseproteins has * To whom correspondence should be addressed. remained elusive. Thus, we reasoned that the isola-

Genetics 119: 579-593 uulv, 1988) 580 G. B. Leicht and J. J. Bonner tion of mutations in the genes encoding these proteinsassociated with this class. If this classwas lethal or had might provide someinsight into their function(s). some visible mutant phenotype, a stock was established by However, given that the small hsp genes represent a crossing the ri pp*lTM3Ser or ri pp*/Dp(3;3)MS4male sibs to TM3SbITM6B females. Once established, all stocks were gene family (INGOLIAand CRAIG1982; SOUTHGATE, retested by backcrossing to Df(3L)ACI. AYMEand VOELLMY 1983;AYME and TISSIBRES1985), Although the vast majority of the mutagenized chro- we realized at the outset that mutations in any one mosomes were of the genotype ri pp, a group of approxi- of the genes might not confer a mutant phenotype matelB 300 EMS-treated chromosomes of thegenotype and that a deletion of the entiregene cluster may be Adhh "Zfm(3)7 were also screened for lethality or visible defects over Df(3L)ACI. Adhh6'" is an insertion of an to required do so. hp70-Adh fusion gene at cytological location 61C (BONNER This paper reports the results of our mutational et al. 1984); the presence of this insertion was irrelevant analysis of the 67A2 to D 11- 13 subinterval and pre- for the purposes of these screens. liminary characterization of the mutations obtained. Isolation of recessive lethal mutations in the 66F3-67B1 This analysis has revealed that (1) this region of the subinterval: Df(3L)29A6,a DEB-induced deletion of 66F3- 67B1 isolated in the above screens, was used to screen for chromosome contains a minimum of 24 essential loci, additional lethal mutations within the 67A2-B 1 subinterval. many of which are required for imaginal develop- These screens were essentially identical in design to those ment, and (2) the small heat shock protein genes are using Df(3L)ACI except that males of the genotype ri e functionally equivalent. were mutagenized (0.007 M DEB). In addition, the F2 generation was subjected to a 36" heat shock during the MATERIALS AND METHODS course of larval or pupal development. The heat shocks were administered in two ways: the first method involved Fly stocks and culture conditions: Df(3L)ACI is a dele- immersion of the vials into circulating waterbaths for 2 hr; tion extendingfrom 67A2 to 67D11-13 generated by the second involved placing trays of vials into an incubator ADELAIDET. C. CARPENTER (unpublished results).TM6B is for 5-6 hr. Putative lethals were backcrossed toDf(3L)29A6 a newer version of the TM6 balancer chromosome that and examined for lethality at both 22" and following a 36" carries the dominant marker Tb (CRAYMER1984). M(3)i55 heat shock. Balanced stocks were established for those is a mutant allele of the M(3)ilocus (MORATAand RIPOLL which retested positively (ie., died over the deletion). Each 1975).Hsp28""is an hp28 mutation resultingfrom insertion stock was subsequently crossed to Df(3L)ACI to identify of a defective P element into the 5' regulatory sequences those mutations which mapped in the region of overlap of the hp28 gene that disruptsits inducibility by heat shock between these two deletions. (EISSENBERGand ELGIN 1987). 9500.1 is a transformant Complementation analysis: Inter se complementation stock that carries wild-type copies of hsp28, hp23 and gene crosses were performed among all DEB- and EMS-induced 1 on the second chromosome (COHEN andMESELSON 1985). lethals obtained from the F2 screens over Df(3L)ACI and The 9500.1 insertion also carriesa nonfunctional hsp26 Df(3L)29A6. Crosses were done between mutations bal- gene (due to the insertion of bacteriophage A DNA se- anced over TM3Sb, and the progeny were scored for the quences into the middle of the coding sequence) and the presence of Sb' individuals. Typically, two virgin females alcohol dehydrogenase (Adh) gene as a selectable marker. were mated with two males in vials, and these were passed A2.1 is a transformant stock that carries wild type copies as needed. Initially, all crosses were done in both directions. of hsp22 and thexanthine dehydrogenase (rosy) gene Later crosses were done in only one direction. Generally, inserted on thesecond chromosome (KLEMENZand GEHRING on the order of 100 progeny were scored per cross, unless 1986). Other mutations, rearrangements,and balancer it was obvious that the two mutations complemented fully. chromosomes are described in LINDSLEYand GRELL (1968) Recombination mapping: To establish whether the lethal or LINDSLEY andZIMM (1985, 1987). mutations recovered over Df(3L)ACI mapped to the 67A- Unless indicated otherwise, all stocks and crosses were D subinterval or were interacting mutations that mapped maintained at either 22" or room temperature on media elsewhere, the recombination frequency between the mu- consisting of cornmeal, molasses, and agar onto which live tation and M(3)i55was measured. Each mutant stock was baker's yeast was sprinkled. For growing larvae for cytology, outcrossed to M(3)i55and rn~tIM(3)i~~females were col- flies were grown on yeast glucose media (10% yeast, 10% lected. Four to six such females were then mated to several glucose, 1.5% agar, 0.15% Tegasept). Df(3L)ACIITM3Sbmales in vials, and these were generally Isolation of recessive lethal mutations in the 67A-D sub- passed twice. The progeny were scored for the presence interval: Males of the genotype ri pp, aged 2-4 days, were of the M,Sb+ (rnut+,M(3)i+lDf(3L)ACI)recombinant class. mutagenized with either 0.006-0.007 M DEB or 0.0125 M In cases where the mutation in question was lethal over EMS (both purchased from Sigma) according to the method M(3)i5j,the mutation was mapped relative to Df(3L)29A6. of LEWISand BACHER(1968) and mated individually in vials Lethal phase determinations: Lethal phase determina- to four orfive TM3SbITM6B virgin females. (Virgin females tions were done in twoways. Initial determinations were were obtained from a "virginizer" stock in which the males for homozygous individuals. To facilitate identification of carry a Y ;3 translocation bearing the dominant tempera- mutant homozygotes, each of the mutant chromosomes ture-sensitive mutation DTS-4.) Mutagenized males were was balanced over TMGB, which carries thedominant mated with new groups of four or five females for four marker Tubby(Tb). T6 causes the larvae and pupae to be consecutive days. Mated females were transferred at 1-2- much shorterand fatter in appearancethan wild type; day intervals for up to 10 days (or until they ceased to lay thus,mutant homozygotes are easily recognizable from fertilized eggs). F1 (ri pP*ITM3S6 or ri pp*ITM6B)progeny their TM6-bearing sibs. For each of themutant lines, were collected and mated individually to Df(3L)ACI PPI embryos were collected overnight on 20mm x 60 mm TM3Ser or Df(3L)ACI pp/Dp(3;3)MS4. The resulting F2 plastic Petri plates containing standard media streaked with progeny were scored foreither absence of the ri PP'I 5% acetic acid. From these, 100 embryos were picked and Df(3L)ACI pp class or some visible morphological defect transferred to hard agar plates. After 24-36 hr, the un- 67A -D 58 Genetics of 67A-D 1 hatched eggs were counted in order to determine whether acetone were added, and the samples were precipitated at any of the mutations conferred embryonic lethality. The - 20" for 1-2 hr. The samples were pelleted in an Eppen- development of hatching Tb' individuals was monitored dorf microfuge (- 10K, 10-15 min, 4"), resuspended in 20 on a daily basis to determine the stage (larval, pupal, or pl SDS sample buffer, and electrophoresed through 12% pharate adult) at which the majority of these individuals SDS polyacrylamide gels (LAEMMLI1970). The labeling died. For some members of each complementation group, patterns were visualized by autoradiography of dried gels. the lethal period was also determined over Df(3L)ACI or, Two-dimensional gel electrophoresis heat of shock pro- in the caseof those which mapped to 67A2-B1, teins: Brains and imaginal disc complexes (three to five) Df(3L)29A6. For these experiments, 20-25 mutlTM6B fe- were dissected from third instar larvae and transferred to males were mated to several Df(3L)ACI (or Df(3L)29A6) a 100-pl drop of TB1 in a humidified chamber. Then 30- males. Generally, 100-200 embryos were collected, trans- 50 FCi [35S]methioninewere added, and the chamber was ferred to hard agar plates, and the development of the placed on a 38" hotplate (under a Styrofoam hat) for 60- Tb+ (mutlDf) progeny was monitored as described above. 90 min. Labeledtissues were transferred to 100pl SDS For mutations which mapped to the 67A2-Bl subinterval, sample buffer in an Eppendorf tube, boiled for 10 min, lethal phases were determined both at normal growth and precipitated with three volumes of acetone. Pellets temperature (22-26") and following a heat shock. To were resuspended in standard lysis buffer (O'FARRELL1975; administer the heat shock, the plates were placed in a 37" 15 plfbrain and discs complex) made up with pH 3-10 incubator for several hours. ampholytes (Servalyte). Two-dimensional gel electropho- Cytological analysis and in situ hybridization: Larvae resis of the protein extracts was performed according to a were grown at 22" on either yeast glucose media or well- modification of the procedure of O'FARRELL(1975). Iso- yeasted cornmeal molasses media under uncrowded con- electric focusing (IEF) gels were prepared with pH 3-10 ditions. Late third instar larvae were dissected in TB 1 [ 15 ampholytes andthe Nonidet P-40 concentration was in- mM potassium phosphate, pH 7; 80 mM KCl; 16 mM NaCI; creased to 3% for better resolution of the high molecular 5 mM MgCl,; 1% polyethylene glycol 6000 (Carbowax)], weight hsps. RNase treatment of the samples and the pre- and the salivary glands were fixed for several minutes in running steps were omitted. IEF gels were run for 12-16 lactic-acetic orcein for standard chromosome squashes or hr at 1000 V at 4". Standard 12% SDS-polyacrylamide gels for 5 min in 45% aceticacid for in situ hybridization were preparedfor the second dimension. Heat shock experiments. In some cases the larvae were first given a proteins were visualized by autoradiography of dried gels. brief heat shock (30 min, 37") to induce the heat shock To facilitate identification of mutations that affected the puffs. small hsps, the mutant lines (balanced over TM6B) were Chromosome squashes were prepared for in situ hybrid- outcrossed to hp28'", which fails to synthesize hsp28 and ization as described by BONNERand KERBY(1982). Hsp22- also carries a variant of hsp22 relative to the mutagenized and hsp28-specific probes were generated by nick-transla- chromosomes. The m~tIhsp28~"larvae were identified by tion of plasmid subclones J1S3 and T6BH2 (IRELANDet al. their nontubby phenotype. 1982), respectively, with ['HIdCTP (Amersham). The Construction of the 9500.1, A2.1double-transformant probes were prepared and hybridized to the samples ac- stock: A double-transformantstock carrying wild-type cop- cording to the procedure of BONNERet al. (1984). 50,000 ies of hsp28, hsp23, hsp22 and gene1 on the second chro- to 100,000 cpm were applied to each slide, and the hybrid- mosome was generated by recombining two second chro- ization was carried out for 12-16 hr at 65-66". Slides were mosome insertions, 9500.1 and A2.1 (see above), onto the washed, dried, and prepared for autoradiography accord- same chromosome As the first step in the construction of ing to the procedure of BONNERet al. (1984). this stock, the A2.l(ry+)insertion was crossed into an Adh Standard chromosome squashes were viewedby phase null [Adhm6]background: A2.l(ry+);h~p22"~~ th st cu ry506 contrast microscopy on a Zeiss photomicroscope. For the males were crossed to Adhrn6 cn; ry506 females, the heter- in situ hybridization experiments slides were stained with ozy ous female progeny were collected and backcrossed to Giemsa then viewed and photographed with bright-field Adh% cn; yfo6males, and the Adhfn6, AZ.I(ry+)recombi- optics on a Zeiss photomicroscope. nants were identified by double-selection on purine-con- Heat-shocking and labeling of individual embryos:Em- taining food and pentynol vapors. For purine selection, 84 bryos were transferred from a collection plate to a drop of pl of 0.44% purine (Sigma) solution was dribbled onto the TBI in a glass depression slide. After removing as much surface of food vials when the developing progeny were at of the buffer as possible with a flame-drawn pipette, the the first to second larval instar stage. For the latter selection, depression slide was covered with a 22 mm coverslip, sealed 3-6-day-old adults were placed in stoppered vials contain- with water or TB1, placed on a 38" hotplate, and covered ing 100 pl 0.4% 1-pentyn-3-01 (ethyl ethynyl carbinol, with the bottom of a glass Petri dish and a Styrofoam hat. Aldrich) solution, dispensed onto a piece of filter paper, (Under these conditions the embryos receive the equivalent for 90 min. Maleswhich survived both selectionswere of a 36-37°Cshock.) Following a1-hr heat shock, the crossed to Adhf*3/CyO"B pr cn2; ry'SblTM3Ser females to embryos were dechorionated with 50% bleach then washed isolate and balance the Adhfn6 A2.1(ryf)chromosome. In extensivelywith TB1. Individual embryos were carefully the second step of the construction, males of the genotype transferred with a tungsten needle to 5 pl drops of 90% Adhrn6 A2.1(ry+)lCyOnBpr cn2; y'Sbl~506were crossed to TBl/lO% ["Slmethionine (Amersham) in a 64-well micro- 9500.1 (Adh+)pr cnlCyOd pr cn2; ry SblTM3Ser females, titer dish. Each embryo was then disrupted with a sharp female progeny of the genotype Adhfn6AP.l(ry')l tungsten needle. The dish was covered with Scotch tape to 9500.I(Adh+) r cn; rySo6/ry Sb werecollected and back- prevent desiccation and the embryos were labeled for 30 crossed to Adhen6 cn; ry506 males, and the putative 9500.1 min on the 38" hotplate. At the end of the labeling period, (Adh+), AZ.I(ry+)recombinant progeny were subjected to 25 p1 SDS sample buffer(1% SDS;10 mM Tris,pH 7; purine selection as larvae and ethanol selection as adults. 0.1% P-mercaptoethanol) were added to each culture, and For the ethanol selections,3-4-day-old adults were sub- the microtiter dish was covered with fresh Scotch tape and jected to 5%ethanol vapors for 24 hr as described by floated in a 68" waterbath for 15 to 20 min. Samples were BONNERet al. (1984). Males which survived both selections transferred to 0.5-ml Eppendorf tubes, three volumes of were crossed to Adhf*31CyOnB prcn2; ry'SblTM3Ser females 582 B. 582 G. Leicht and J. J. Bonner to isolate and balance the newly generated 9500.1, A2.1 double-insertion chromosome.9500.1, A2.1 I CyOd pr cn2 progeny were identifiedby selection on 5% ethanol vapors. To verify that the recombinants so isolated carried and ex ressed both insertions, several "9500.1 A2.1 "lCyOdpr cn' recombinantlines were outcrossed to Df(3L)29A6/ TM3Sb, "9500.1,A2.1 "I+; Df(3L)29A61ry males and +I CyOnB;Df(3L)29A6/ry females were collected and mateden mse in eggcollection bottles, and the hsp profiles of individual embryos (3-7 hr old) derived from the latter crosswere examined as described above. In general, a minimum of 32 embryos were labeledfor each recombinant line. The expectedclasses andrelative frequencies of embryos from the aforementioned crosses are as follows: three-fourths of the embryos will receive at least one wild- I ACI I DF(3L)67A2:67D11-13 type third chromosome and will synthesize all four small DF(3L)66F3.6781 hsps upon heat shock; one-eighth will be homozygous for Df(3L)29A6 but will fail to receive the 9500.1, A2.1 chro- FIGURE1.-Cytogenetic map of the 67 interval of chromosome mosome and will synthesize none of the small hsps upon 3. The genetic map position and/or cytological location of the heat shock; one-eighth of the embryos will be homozygous previously known genetic loci within the 67 interval are indicated for Df(3L)29A6 and will also receivethe 9500.1, A2.1 at the top of the map. The visible mutation curuoid (cur) is boxed chromosome,and should synthesize hsp28, hsp23, and to indicate that this map position is in question. The two flanking hsp22, but no hsp26 upon heat shock. visible markers hairy (h)and rose (rs)are also included on the map Rescue crosses with 9500.1, A2.1:The ability of the as points of reference. [Map positions were taken from LINDSLEY 9500.1, A2.1 double-transformant chromosome to restore and GRELL(1968), LINDSLEYand ZIMM (1985, 1986) and other viability was determined foreach of the lethal complemen- references cited in the text.] Two deficiencies of the region, tationgroups within the 67A2-BI subinterval. Rescue Df(3L)ACI (A. T. C. CARPENTER,unpublished results) and crosses were performedas outlined in Figure 7.A minimum Df(3L)29A6 (this publication) are diagrammed at the bottom of of 100 progeny was scored for each cross. the map. Correction of errors: A preliminary report of the lethal mutations in the 67A2 to Dl 1-13 subintervalwas published in Drosophila Information Seruace, 64, pp: 68-69. Subsequent other loci along the chromosome. Thus, DEB was complementation and mapping analysls revealed that sev- eral of the allele assignments were in error. Amendments utilized for this specific purpose. Finally, the use of have been made for complementation groups 1(3)67BDj, a combination of would diminish the like- 1(3)67BDn,and I(3)67BDp (see Figure 3). lihood of missing certain classes of mutations due to sequence specifity of either . Altogether approximately 8700 chromosomes were RESULTS screenedusing DEB as themutagen and another Isolation of recessive visible and lethal mutations 7800 were screened using EMS as the mutagen. A in the 67A-D subinterval: Using a deletion extending total of 108 mutations which exhibited lethality over from 67A2 to 67Dl1-13, Df(3L)ACI (Figure l), we Df(3L)ACI were recovered (see Table 1 for a break- carried out a series of standard FP screens for both down of the mutations).Interestingly, 13 of these recessive visible and lethal mutations throughout this mutations, while lethal over the deletion, were viable region of the third chromosome (see MATERIALS AND as homozygotes. No visible mutations were recovered METHODS for descriptiona of the mutagenesis with eithermutagen. Notably, the mutation fre- scheme). Two different mutagens were used in these quency obtained with the two mutagens was roughly screens: EMS, a potent alkylating agent, and DEB, a equivalent (0.0061 with DEB us. 0.007 with EMS). mutagen reported to generate both intra- and inter- Complementation and mapping analysisof reces- genic deletions (SHUKLAand AUERBACH 1980; OLSENsive lethal mutations:In orderto (1) assign the lethal and GREEN1982; REARDONet al. 1987). These two mutations isolated over Df(3L)ACI to complemen- mutagens were chosen for a numberof reasons. First, tation groups and (2) determine whether any of the we wished to saturate the 67A-D subinterval with a mutations were actually deletionsspanning several variety of point mutations as a means of genetically loci, inter se complementation crosses were performed characterizing this region of the genome. EMS was among the mutant lines. (The 13 homozygous viable chosen for this purpose because it is known to gen- lines were not included in this analysis since allelic erate point mutations with high efficiency. Second, relationships would have been difficult to establish. since one of the goals of our analysis of the 67A-D In fact, two of the lines, both of which were weakly region was toobtain mutations of the small heat viable as homozygotes and maintained as balanced shock proteingenes, we wished touse a mutagen stocks over TM3, were initially included in the com- which could potentially generate a setof overlapping plementation crosses and were found to complement deletions encompassing this gene cluster. Such over- all of the recessive lethal mutations.) In conjunction lapping deletions would also be useful for ordering with the complementation analysis, mapping crosses Genetics of 67A-D 583

TABLE 1 A. Mutation in 67hD Subintomi Summary of screens for recessive lethal and visible mutations in the 67A2 to 67Dl1-13subinterval

I. Results of DEB screensa Total no. of chromosomes screened 8692 No. of lethal phenotypes over Df(3L)ACI 53 Homozygous lethal 48' Homozygous viable 5 No. with visible phenotypes over Df(3L)ACI 0 Mutation Frequency 0.0061 11. Results of EMS screens' Total no. of chromosomes screened 7814 No. with lethal phenotypes over Df(3L)ACl 55 8. Mutation not in 67A-DSubintewai Homozygous lethal 47 W3Y +

Homozygous viable 8

No. with visible phenotypes over Df(3L)ACI 0 Mutation Frequency 0.0070 + M3Y 1: W(3L)ACl '? x "." "" ' ....- * DEB (0.006-0.007 M) was used. - + + msb One of the lethal mutations was cytologically identical to Df(3L)ACI. A + + wy f EMS (0.0125 M) was used. ". mp c - . -...... a . . .- .-.. .. 1 a -a werecarried out to lethalverify thatthe mutations did viable indeedmap to the 67A-D interval and were not Expect highfrequency interacting mutations located elsewhere in the gen- FIGURE2.-Mapping strategy used to determine whether the ome. This was an important fact to establish for two lethal mutations map to 67A-D. If the lethal mutation (designated reasons. First, Df(3L)ACI uncovers the Minute locus, here as an asterisk) maps to 67A-D, it will be tightly linked to M(3)i, at 67C and Minute loci are known to exhibit M(3)i. Therefore, in crosses of */M(3)is5females to Df(3L)ACI lethal interactions with loci located elsewhere in the males, few or no wild type (+ +) recombinants will be recovered genome (LINDSLEYand GRELL1968; MOSCOSODEL over Df(3L)ACI (A). If, on the other hand, the lethal mutation maps outside of the 67 region (for example, to the right arm), PRADOand RIPOLL1983; B. G. LEICHT,personal wild-type recombinants will be recovered at a higher frequency observations). Secondly, Df(3L)ACI is a very large (B). deletion, encompassing over 40 chromosome bands. In general, such large deletions lower viability be- units. M(3)i mapsto roughly the middle of this cause of aneuloidy for the loci containedtherein. region; therefore, any mutations with recombination Thus, Df(3L)ACImight be lethal in combination frequencies of less than 0.02-0.03 shouldreside with nonoverlapping deletions and/or mutations that within this subinterval. also lower viability. While such interacting mutations The results of the mapping crosses for the DEB- may beinteresting in their own right, we did not induced lethals are presented in Table 2. It is clear wish to study mutations that were not actually within from thesedata that approximately one-fourth of the 67A-D region. thesemutations do not map to the 67A-D region, The mapping scheme we employed involved mea- exhibitingrecombination frequencies of 0.05 or suring the recombination distance between the lethal greater. Some of these "interacting" mutations reduce mutation of interest and a mutation known to map the viabilityof the fly considerably, so it is not within the 67A-D ~ubinterval"M(3)i~~(see Figure surprising that they are lethal in combination with 2). M(3)Fj5, like the recessive lethal mutations isolated Df(3L)ACI. Many of thesemutations also display in our screens, is lethalover Df(3L)ACI. If one ratherunusual complementationbehavior as well. generates females that are heterozygous for the mu- First of all, the complementation patterns for some tation of interest and M(3)P (ie., rn~t*/M(3)2~~) andof these mutations appear to be random; e.g., they crosses these females to Df(3L)ACI/TM3 males, only fail to complement certain members of other com- rnut+M(3)zfrecombinants will be viable over the plementationgroups while fully complementing deficiency. If the mutation is closely linked to M(3)2, other members of the same group. Second, all of then few recombinant progeny are expected to be these interacting mutations appear to represent dis- observed (Figure 2A). If the mutation is not tightly tinct loci; none fall into the same complementation linked, a higher frequency is expected (Figure 2B). group.This is in contrastto those DEB-induced Based on the mappositions of the two flanking visible mutations which do map to the 67A-D subinterval, markers h (26.5) and TS (35.0), as well as the number many of which have two or more members. There- of bands in the 67 interval, we estimate that the 67A- fore, instead of mapping each of the EMS-induced D subinterval corresponds to approximately 5 map lethals in this samefashion, we assumed that any 584 B. G. Leicht and J. J. Bonner

TABLE 2 Mapping data for DEB-induced lethal mutationsrecovered over Df(3L)ACI

Parental classes Recombinants Parental classes Recombinants Mutant Mutant line mutlTM3SbM(3)255/TM3Sb mut+, hf(3)2+/ACI line mutlTM3SbM(3)t5’ITM3Sb mutt, M(3)i’lACI - T d5A? 41 45 19 (0.442) dl 4A 7 54 75 1 (0.016) d?A2 20 12 20 (1.250) d45C2 169 143 1 (0.006) dlOAl 10 9 14 (1.474) dl OA5 116 96 1 (0.009) dl 4A5 170 140 2 (0.013) dl 5A3 32 28 22 (0.733) d49A4 184 105 0 (0.000) d5A 1 200 147 0 (0.000) d4 7B5 143 150 0 (0.000) dl OA? 179 125 1 (0.007) d7A6 101 74 1 (0,011) d29A6 183 152 0 (0.000) d49A5 49 61 0 (0.000) d? 7AI 58 74 0 (0.000) d7A5 220 196 1 (0,005) d44A2 87 60 0 (0.000) d4D2 106 80 0 (0.000) d48A6 60 63 0 (0.000) dl 7A6 84 51 0 (0.000) d50AI 146 122 7 (0.052) d?6A 7 53 64 0 (0.000) d58AI 17 48 d54A 7 110 163 3 (0.022) 20 (0.615) d?8A5 79 114 1 (0.010) d33A? 213 144 0 (0.000) d?8B5 101 127 3 (0.026) d48A? 71 71 18 (0.253) d58A 7 128 122 2 (0.016) d42A4 86 41 54 (0.850) d4A3 50 59 0 (0.000) d5IB2 52 78 0 (0.000) d52A2 71 54 29 (0.464) d8AI 76 82 1 (0.013) Parental classes Recombinants dl 4A3 84 85 1 (0.012) Mutant line mutlTM3Sb or 1(3)d29A61TM3Sbmul+, 1(3)+/ACI d49A? 93 89 1 (0.011) d7A? 80 91 1 (0.012) dlOA4 129 15 (0.233) dl 9A? 135 118 3 (0.024)7A5 d2 69 25 (0.725) d2?A? 118 115 0 (0.000) d44A5 112 11 (0.196) d?5A6 96 55 0 (0.000) d48B6 187 19 (0.203) dl IA5 116 95 2 (0.019) L Crosses were performed as described in MATERIALS AND METHODS.Mutations within the same complementation group are grouped together. The apparent recombination distance between the mutation and M(?)i locus is bracketed by parentheses. To calculate the recombination distance, we used the equation “2(no. of recombinants)/no. of parentals,” which takes the lethality of certain progeny classes and the indistinguishability of the recombinantlTM3 class from the parentaVTM3 class into account. The apparent recombination values of greater than 50% for some of the mutant lines is attributable to greater vigor of the wild type recombinants relative to their non- recombinant sibs. Mutant lines d5A?, d?A2, dlOAI, 15A?, and d58A1, in particular, are only weakly viable as heterozygous stocks. Note: the four mutantlines at the bottom right are lethal over M(?)Zy6 and were mapped relative to l(?)d29A6, one of the lethal mutations shown to map to 67A-D. mutation that fell into a complementationgroup with of the DEB-induced lines are cytologically normal. two ormore members anddid not show random Thus, ifDEB doesgenerate deletions, most are patterns of complementation most likely mapped to intragenic. Secondly, these mutations define 22 dif- 67A-D. Thirty-two of the EMS-induced lethals met ferent lethalcomplementation groups within the these criteria, and thuswere assumed to be mutations 67A2 to Dl 1-13 region. If one also includes the M(?)i within the 67 interval.The remaining mutations were locus, for which no new mutations were recovered, mapped, and six of these were found to map else- thereare at least 23 vitalloci within this region. where (data not shown). Finally, the distributions of recessive lethal mutations Figure 3 is asummary of thecomplementation obtained with the two mutagens are roughlythe analysis among those recessive lethal mutations which same. There does not seem to be any site specificity map to the 67A2 to Dl 1-13 subinterval on the basis for either mutagen, at least on a gross level. of recombination. Several conclusions can be drawn Identification of d29A6 as a deletion of the small from this analysis. First, of the 74 lethal mutations heat shock gene cluster: Complementation analysis isolated in this region, only two (d29A6 and e146C4) among the DEB- and EMS-induced lethals indicated behave like deletions, and only one of these (d29A6) that d29A6 was a deletion spanning several comple- was induced withDEB. All theothers behave like mentationgroups. Cytological examination of the apparent point mutations. Indeed, cytological anal- polytene chromosomesof d29A6/TM? larvae notonly ysis revealed that, except for d29A6 (see below), all confirmed the presence of a deletion within the 67 Genetics of 67A-D 585 region,but revealed that the proximal breakpoint ...... was at 67B1, the site of the small hsp gene cluster. The distal breakpoint was located at 66F3. Thus, this deletion removes 14 chromosome bands (see Figure 1). In order to determine whether small the heat shock geneswere deleted in line d29A6, we performed three different assays for their presence. First, po- lytenechromosome squashes were preparedfrom heat-shocked salivary glands of d29A6 heterozygotes, and the d29A6 homolog was examined for the pres- ence of the characteristic 67B heat shock puff. This analysis revealed that the puff was missing in the d29A6 chromosome, and suggested that most, if not all, of the cluster was deleted (data not shown). To demonstrate that the entirecluster was in fact deleted, in situ hybridization and proteinlabeling experiments were performed. In the former, polytene chromo- somes from d29A6 heterozygotes were hybridized with 3H-labeled DNA probes specific for hp28 and hp22, the two flanking genes of the cluster. If the entire cluster was missing, neither probewas expected to show any hybridization to thed29A6 homolog, and this was indeed the case (data not shown). Finally, the absence of all four of the small heat shock protein genes was demonstrated by one-dimensional gel elec- trophoresis of [ 35S]methionine labeled proteins from heat-shocked embryos (5-9 hr of development) de- rived from the d29A6ITM3 line. It was expected that one-fourth of the embryos would be homozygous for the deletion and, if missing the entire small hsp gene cluster, would not synthesize any of the small hsps. As the autoradiogram in Figure 4 shows, this expec- tation was met. Having verified that d29A6 was deficient for the small hsp gene cluster, we used this chromosome to screen for additionallethal mutations within the

FIGURE3,”Summary of complementation and mapping data for lethal mutations isolated over Df(3L)ACI. Deletions are indi- cated by the heavy horizontal lines; the various complementation

-h groups (andmutations included therein) are listed below. The two g$g small deletions, Df(?L)29A6 and e146C4, were isolated as lethal z2:: ocI..o mutations over Df(3L)ACI and were identified as deletions by ...... their complementation behavior. The breakpoints of Df(3L)29A6 were assigned by cytological examination of the polytene chro- mosomes. The breakpoints of e146C4 have not been defined as this deletion is too small to detect cytologically. Complementation groups uncovered by both Df(?L)ACI and Df(3L)29A6 have been designated as 1(3)67Aa to 1(3)67Ae;complementation groups un- covered by only Df(3L)ACl have been designated as 1(3)67BDato 1(3)67BDr. (Note that complementation group 1(3)67BDo corre- sponds to the M(3)i locus.) DEB-induced mutations are preceded by a lowercase “d”; EMS-induced mutations are preceded by a lowercase “e.” Mutant alleles enclosed by brackets appear to represent weak allelesin that they exhibit partial complementation with other members of the complementation group. It should be ...... >...... 1. noted that the order of the complementation groups within the two sub-intervals defined by Df(3L)ACI and Df(3L)29A6 has not yet been determined; thearrangement shown here is strictly arbitrary. 586 B. G. Leicht and J. J. Bonner

1 2345678 W3L)ACl (67A2 1067011-I3 66F3 6781 I Gf(3L)ZllAS Cl46C4

~ 113I67Aa 1(3)67Ab l13167AC Il3J67Aa KJI67Ae 113J67AI d17~A3618501, """ dZQ434, dZ48112 dJ7AI &IC 67884 e15X4 d72A2 e17 e7383 e76AI dl89QZ eIC6C.3 bz36Al dl/dE5 6232A2 El I FIGUREj.-Summary of complementation analysis among mu- tations in the 67A2 to 67B1 subinterval. Lethal mutations E1.I and e17 are EMS-induced mutations provided by GLES COLLIER - hsp28 - hsp26 and KATHLEES MATTHEW,respectively. The mutations which map - hsp23 to the 66F3-67A1 subinterval-the region unique to Df(?L)29A6- - hsp22 have not been placed into complementation groups.

67A2-B 1 region complementation groups or repre- sented newly identified loci, inter se complementation crosses were performed among these mutations and FIGURE4.-Protein synthesis profiles of individual heat-shocked the nine previously identified mutations. This anal- embryos from the d29A6/TM? line. Embryos ranging in age from ysis revealed that five of the new lethals were mem- 5 to 9 hrs of development were collected, heat-shocked for 1 hr, bers of previously identifiedcomplementation andthen labeled with ["Slmethionine. Labeled proteins from groups, while the sixth (e17) appeared to define a individual embryos were separated by electrophoresis through a 12% SDS-polyacrylamide gel and viewed by autoradiography. new complementation group, thereby bringing the Shown hereis an autoradiogram of the labeled proteins from eight number of identified essential lociin the 67A2-Bl embryos.One-fourth of the embryos (lanes 1 and 2) fail to region to six. Figure 5 shows an enlargement of the synthesize the small hsps. (Note: the variable expression of hsp28 complementation groups in the 67A2-Bl region and and hsp22 is typically observed with embryos.) includes the six new mutations. Lethalphase determinations: In order to begin 67A2-Bl subinterval (the region of overlap between characterizing the different lethal complementation d29A6 and Df(3L)ACI), with the aim of (1) saturating groups identified above, the lethal periods were this smallerregion with lethal mutations and(2) determined for members of each (see MATERIALS AND isolating mutationsthat affect the small hspgene METHODS for experimental details). Because of the cluster specifically. We again performed standard F2 large number of complementation groups, the mu- lethal screens, but subjected the developing F2 prog- tations were simply classified as embryonic, larval, eny to a 36" heat shock, reasoning that mutations in pupal, or pharate adult lethals; no attempt was made the small hsp genes might notbe lethal in the absence to define the exact point in development at which of a heat shock. DEB was again used as the mutagen each died. The results of this analysis are presented in the hope that we might recover another deletion in Figure 6. [It shouldbe noted that these lethal spanningthe small hspgene cluster, as we felt a phasedeterminations are for mutant homozygotes deletion of the cluster was more likely to produce a (black circles) as well as trans-heterozygotes carrying lethal phenotype than simple point mutations. the mutation over either Df(3L)ACI or Df(3L)29A6 Out of approximately 6000 chromosomes screened (stippled circles)]. for lethality over d29A6 (which will henceforth be Several major findings cameout this analysis. First, referred to as Df(3L)29A6), four new lethal muta- many of the EMS-treated mutant chromosomes ap- tions in the67A2-Bl subinterval were recovered. pear to carry accessory lethal mutations with earlier (Four other lethal mutations which mapped to the lethal phases (ie., mutant homozygotes die earlier in 66F3-67A 1 region were also recovered.) Backcrosses development than themut/Df heterozygotes)-a find- to Df(3L)29A6 and retesting for lethality at both 22" ing which was notunexpected for this mutagen. and after a 36" heat shock revealed that all of these While this is also true of some of the DEB-treated were lethal at both temperatures; thus, none were chromosomes, this mutagen appears to generate pre- strictly heat shock lethals. In addition to thefour dominantly single-site mutations. In general, mutant new DEB-induced lethals in the 67A2-Bl subinterval, homozygotes have the same lethal phase as each two new EMS-induced lethals which mapped to this other and as individuals carrying the mutation over region were isolated in independent screens by KATH- the deficiency. Secondly, many of the lethal comple- LEES MATTHEW and GLES COLLIER(unpublished mentation groups have late lethal periods-late larval results). to young adulthood. Finally, several of the comple- To determine whether these six new lethal muta- mentationgroups (e.g.,1(3)67BDb and 1(3)67BDh) tions (d174B.5, d189B2, (1232A2, d236A1, el7, EI.1) may represent complex loci in that some alleles have were mutations within the previously identified early lethal phases and others have late lethal phases. Genetics of 67A-D 587

e61C I 0PA l(3)67Ab r76A 1 0 PA d236A 1 I op* IK3b67A.s e72A2 0 0

~ 1(3)67Ad e7W.i. 0 0 e 146C4 .o 1(3)67A@ 1) 152C4 FIGURE6.--Summary of lethal dlW2 0 I phase analysis. All lethal phase de- terminations were done at room tem- perature (22-26”) except for com- plementationgroups 1(3)67Aa- 1(3)67Ae,which also were examined following a 36-37” heat shock. No differences in the lethal periods were noted at the two temperatures, how- ever. Lethal periods of mutant homozygotes are indicated by the black circles;lethal periods for trans- heterozygotes carrying the mutation over one of the two deficiencies are indicated by the stippled circles. “PA indicates that at least some of the mutantindividuals survive to the pharate adult stage. a 78B4/Df(3L)ACI individuals survive to early adulthood under the conditions of this experiment and have somewhat smaller and rougher eyes than wild type. Some individuals also have held backwings. Viable adults have not been observed under standard culture conditions in vials. These two mutations appear to be somewhat leaky in that 10-15% of the mutant individuals will de- velop to adulthood and eclose. In the case of e7584, this apparent leak- iness pertains to e75B#/Df(3L)ACI individuals only; e7584 homozygotes die as embryos, presumably due to an accessory embryonic lethal mu- tation. The mutant individuals die either shortly after eclosion (e94DI) or within the first day or so there- after (e140BI). 588 B. G. Leicht and J. J. Bonner Alternatively, this variance could be due to differ- to determine if they diedat anearlier stage of ences in severity of themutant alleles. Additional development relative to unshocked sibs of the same analysis will be required to address this issue. genotype. No difference in lethal phase was noted While most of these lethal complementation groups for any of the six complementation groups. Thus, do not have distinctive phenotypes, at least at the each was able to mount a normal heatshock response level of examination used here, two of the comple- by this criterion. mentation groups do have rather intriguing pheno- We next examined the profile of small hsps syn- types. Complementation group 1(3)67BDrn, repre- thesized by each of the mutant lines by two-dimen- sented by the mutation d47B5, is one of several with sional gel electrophoresis. To facilitate identification a late larval lethal phase. However, unlike the others, of mutations affecting the small hsps, we outcrossed which simply develop to a certain pointand then die, each to a stock, hf128~~',which is deficient for hsp28 d47B5Id47B5 or d47B5/Df(3L)ACl individuals arrest and carries a charge variant of hsp22 relative to the at the late third larval instarstage. Themutant parentalchromosome (~ipp or ri e) on which the individuals remain as mature third instar larvae for mutations were induced. Because hsp28 and hp22 up to several weeks, before eventually dying as pseu- are the two flanking genes of the cluster, deletions dopupae. Attempts to restore normal development coming in from either end of the cluster would be by the administration of exogenous @-ecdysone,the readily identifiable. For example, if a deletion of the insect moulting hormone, revealed that this pheno- proximal end of the cluster (i.e., the end flanked by type was not due to hormone deficiency. Subsequent hp28) had been generated, the rn~tlhp28~~'hetero- dissection of the mutant larvae revealed that they zygotes would lack hsp28, whereas if a deletion of lack imaginal discs, accounting for their inability to the distal portion of the cluster had been generated, undergo metamorphosis.Complementation group the rn~tIhp28~~'heterozygotes would have only one 1(3)67Ab,represented by mutations e61C, e76A1, and spotfor hsp22. (The orientation of the small hsp d236A1, exhibits abnormal blackening of the pupar- genes with respect to the telomere of chromosome ium as well as some unusual cuticular defects. The 3L is hp22-h.sp26-genel-hsp23-hsp28 as determined by latter are variable; some mutant individuals have no in situ hybridization of hp22 and hsp28 probes to obvious cuticular defects while others have a belt of polytenechromosomes of heat-shockedlarvae; cuticle around the midsection. Interestingly, despite LEICHT1987). In addition to identifyingdeletions, the abnormal darkening of the puparia, the devel- this analysis would also serve to identify any mutants oping imagos within do not exhibit abnormal pig- with small hsps of altered charge or size. mentation and some, in fact, develop into fully pig- Examination of the two-dimensional profiles of mented pharate adults. None of these pharate adults [ 35S]methionine-labeled proteins from heat-shocked eclose, however. brains and imaginal discsof rnutlh~p28"~'larvae re- Tests to determine whether any of the 67A2-Bl vealed essentially no differences relative to the pa- lethals represent mutations of the small hsp genes: rental line (data not shown). In every case, all of the In order to determine whether any of the mutations small hsps appeared to be synthesized (ie., the pa- that mapped to the 67A2-B 1 subinterval represented rental hsp28 and hsp22 variants were present and mutations and/or deletions of the small hsp genes, the amounts of hsp26 and hsp23 appeared normal) three different assays were performed. First, each of and there were no obvious shifts in mobility of any the mutant lines was heat shocked early in develop- of these proteins. By this criterion, each of the six ment to determine whethera heatshock would result 67A2-B 1 lethal complementation groups synthesizes in precocious onset of lethality. Secondly, the pattern a normal complement of small hsps. of hsps synthesized by each was examined by two- While the foregoing two-dimensional gel analysis dimensional gel electrophoresis to determine if any ruled out thepossibility that any of the mutants carry differences could be found relative to the parental small deletions of the small hspgene cluster, this line. Finally, each line was crossed to a transformant analysis didnot rule out the possibility thatpoint stock carrying wild type copies of three of the four mutations of the small hsp genes, which result in no small hsp genes on the second chromosome to address chargeor size changes of theproteins, had been whether introduction of wild-type copies of the genes generated. Thus, as a final means of determining would restore viability. whether we had isolated mutations within the small To investigate whether any of themutant lines hsp gene cluster,we performed "rescue" crosses with would die precociously if given a heat shock, each a transformant stock of flies, 9500.1, A2.1, carrying was backcrossed to Df(3L)29A6lTM6B,embryos were wild-type copies of hp28, hsp 23, hp22 and gene 1 collected and allowed to develop to first instar, and inserted into the second chromosome (see MATERIALS these were then heat shocked (36-37", 4-5 hr). The AND METHODS for a description of this stock). In other mutlDf(3L.)29A6 individuals, identified on the basis words, we asked whether introduction of wild-type of their nontubby phenotype, were simply observed copies of the small hsp genes would alleviate the Genetics of 67A-D 589

A. Rescueof muWmB B. Rescue of muVDf(3L)29AG

/ 9500 1prmAZ.l B'ri A'd (-jl(-jl x L %OO.lpcnA21 Gf(3L)29A6. ri + Y + TM3Sbor TMGB 99 viable? + mu( ri

1 Score la (he presence d radius incomplelus. non-Stubble progeny %5W.IpcnA21 A' d - viable? + 8' n

A'.W = dllerenl &re la the presence d radlus mcanpletus. non-StuMe progeny

FIGURE7.-Outline of rescue crosses with the 9500.1, A2.1 double transformant stock. For complementation groups with more than one member (e.g., 1(3)67Aa, 1(3)67Ab, and 1(3)67Ad), the scheme outlined in panel A was used. For complementation groups with a single member (e.g., 1(3)67Ac, 1(3)67Ae, and 1(3)67Af), the scheme outlined in panel B was used. In both cases, the presence of progeny with the radius incompletus phenotype is diagnostic for rescue since this marker is carried by the parental chromosomes from which Df(3L)29A6 and the various lethals were derived.

TABLE 3 Df(3L)29A6).The results of these crosses, which are presentedin Table 3, revealed an inability of the Results of rescue crosses with 9500.1, A2.1 double transformant stock transformant chromosometo rescue the lethality associated with any of the six 67A2-Bl lethal com- A. Rescue of lethality of trans-heterozygotes (mutA*lmutB*) plementation groups. No. of No. of The results of the foregoing experiments allowed mutA*lBal, mutB*lry, mutA*lmutB* us to conclude, at the very least, that none of the mutA*lmutB* or VIBal progeny progeny 67A2-B 1 lethal complementation groups represent d37AI Id1 74B5 245 0 lesions in the genesencoding hsp22, hsp23 and d37AlldZ32A2 219 0 hsp28. (We knew at that outset that flies which are e73B3ldI 7485 254 0 homozygous for amutation of hsp28, h~p28'~',are e73B3ld232A2 252 0 viable at both normal and heat shock temperatures; e76All236Al 102 0 thus, we did not expect any of the lethal mutations e152C4ld189B2 306 0 to represent lesions in that gene.) Although it is B. Rescue of lethality of mutlDf(3L)29A6 formally possible that some of the mutants carry a No. of No. of in hsp26, we believe this is unlikely mutlln(3L)P,Df(3L)29A6lTM3, mutlDf(3L)29A6 for reasons presented in theSUMMARY AND DISCUSSION. Mutant or ln(3L)PITM3 progeny progeny

e72A2 114 SUMMARY AND DISCUSSION el 7 120 Genetic characterization of the 67A2-Dll-13 e78B4 214 subinterval: In carrying out the foregoing genetic Mutations within the same complementation group are analysis of the 67A2 to D 1 1-13 subinterval of D. grouped together. melunoguster,we had two major objectives: first,to bettercharacterize this region of the Drosophila lethality associated with any of the mutations. The genome, andsecond, to isolate mutations in the 67B 1 rescue crosses are outlined in Figure 7. Because the small hsp gene cluster specifically. With respect to lethal phase analysis had revealed that many of the our first goal, this analysis has been quite fruitful. mutants carry accessory lethal mutations that could Out of over 16,500 chromosomes screened for visible mask the ability of the transformant chromosome to or lethal phenotypes over Df(3L)ACI, we recovered rescue lethality, we assayed for rescue of mutant a total of 74 (33DEB-induced, 41 EMS-induced) trans-heterozygotes (i.e., mutAlmutB or mutl independent recessive lethal mutations which map to 590 B. G. Leicht and J. J. Bonner the 67A-D region onthe basis of recombination these are several which map in or near the 67 region, mapping and complementation behavior. No visible suggesting that we may have isolated a new allele of mutations were recovered. Among the DEB-induced one of them. However, tests for allelism revealed that mutations was smalla overlappingdeletion, d47B5 is not allelic to any of the previously identified Df(3L)29A6, extendingfrom 66F3 to 67B1. Using discless mutants, and therefore defines yet another this deletion, we screened an additional 6000 chro- locus involved in disc development. mosomes and recovered another four DEB-induced Complementation group 1(3)67Ab (e61C, e76A1, lethal mutations within the 67A2-B 1 subinterval.Two d236Al) exhibits abnormal blackening of the pupar- additionallethal mutations (both EMS-induced) ium and variable cuticular defects. This phenotype within 67A2-Bl were provided by KATHLEENMAT- is suggestive of a defect in tyrosine metabolism as THEWS and GLEN COLLIER. Altogether, these80 mu- many of the oxidation products of tyrosine play an tationsdefine 23 distinct lethal complementation intimate role in both the sclerotization and pigmen- groups-six thatmap to the 67A2-Blsubinterval tation of the cuticle that occurs at pupariation and and 17 that map to the 67B2 to Dl 1-13 subinterval. adult eclosion (POODRY1980). Among the major If one includes the M(3)ilocus (for which we did not oxidation products of tyrosine are N-acetyldopamine recover any new alleles), there are, at the least, 24 and 3,4-dihydroxyphenylalanine(dopa), which are vital lociwithin the 67A2 to D 1-11 3 subinterval.Using ultimately convertedto sclerotin and melanin,re- an equation that approximates the Poisson distribu- spectively. Excess melanin levels have been demon- tion (no = n:/2n2, where no represents the number strated in the puparium of a black mutant of the of loci for which no mutantalleles have been isolated, blowfly,Lucilia cuprina (HACKMANand GOLDBERG nl representsthe number with onemutant allele, 1968),and are thought to be responsible forthe and n2 representsthe number with two ormore darkening of ebony and black mutants of D. melano- alleles), we estimate that there are another three or gaster (HODGETTSand KONOPKA1973; HODGETTSand four vitalloci which remain to be identified. With CHOI 1974). In Drosophila, the degree of melaniza- respect to the 67A2-B 1 subinterval,calculation of the tion of the cuticle is affected by the production and Poisson distribution predicts that one or two of the incorporation of p-alanine, which is thought to form remaining loci lie in this region. Thus, by this crite- cross-links with indole derivatives of dopamine and rion, we have nearly saturated this region with lethal therebyreduce polymerization of these derivatives mutations. into melanin (HODGETTSand KONOPKA1973). Thus, Lethalphase analysis of each of the 23lethal it is conceivable that the unusual blackening of the complementation groups has shown that they have puparium (and associated cuticular defects) of com- lethal periods ranging from embryonicto early adult- plementationgroup 1(3)67Ab is aresult of either hood. The majority, however, have latelethal pe- inappropriate levels, incorporation, or cross-linking riods-late larval or beyond-suggesting that this of indole derivatives of tyrosine in the larval cuticle, region of thethird chromosome contains a fairly leading to excessive polymerization of these deriva- large number of loci that are specifically required tives intomelanin. Examination of the levels of for imaginal development. Of the various late lethal tyrosine and its derivatives in these mutants will be complementationgroups, 1(3)67Ab and 1(3)67BDm needed to ascertain whetherthis hypothesis is correct. are of particular notein that the terminal phenotypes The other pupal lethal complementationgroups haveprovided some clues as to what theprimary have no distinctive phenotypes except that, in gen- defect might be. eral, they fail to undergo normal differentiation of Complementationgroup 1(3)67BDm (d47B5)ap- imaginal structures.Unlike 1(3)67BDm,this is not due pears to represent a locus required for normal ima- to lack of imaginal discs; rather, the discs form but ginal disc development. Mutant homozygotes develop do not give rise to differentiatedadult structures. quite normally until the endof the third larval instar, This is the expected phenotype for mutations within at which time they undergo developmental arrest, ecdysone-responsive genes. Notably, studies of ec- remaining as roaming third instar larvae for up to dysone-responsive loci by BONNER and PARDUE(1976) several weeks before dying as pseudopupae. Exami- indicated that there is an ecdysone-inducible locus at nation of these mutant larvae has revealed that they 67B 11. Thus, oneof the pupal lethal complementa- lack imaginal discs. The genetic studies of SHEARN tion groups may very well represent a mutation(s) of and coworkers have shown that there are many loci this locus. scattered throughout the genome that are required The recovery of only lethal mutations out of over for normal disc development(SHEARN et al. 197 1; 20,000 chromosomes screened is noteworthy in two SHEARN andGAREN 1974).On the third chromosome respects.First, as mentionedearlier, the recessive alone, a total of 52 lethal complementation groups wing mutation curuoid had been reported to map to with either discless or small disc phenotypes have approximately the middle of the 67 interval (LINDSLEY been isolated (SHEARNand GAREN1974). Among and GRELL1968). If this locus is contained within the Genetics of 67A-D 59 1 67A-Dsubinterval, we shouldhave isolated new cluster. While we cannot rule out the possibility that alleles. The fact that nonew cur alleles were recovered none of the mutations is a lesion within hsp26, we suggests that this locus lies outside this region. In feelthis is unlikely. As stated in the Introduction, fact, we have found that curlDf(3L)ACl individuals the small hsps comprise a gene family and are likely are wild type in phenotype. Secondly, the lack of to have similar, if not identical, functions. Indeed, recovery of visible mutations inthe 67A-D subinterval genetic analyses of a number of other gene families may indicate either of two things: (1) this region of strongly support the view that, in general, members the third chromosomecontains predominantly,if not of the same genefamily are functionally homologous. solely, essential genes, or (2)this region contains loci A major case in point is the Drosophila hsp70 gene which, if mutated, produce no or only very subtle family. In most strains of D. melunogaster, there are changes in visible phenotype due to either the pres- five genes encoding hsp70, two at 87A7 and three at ence of other genes that provide the same function 87C1 (ISH-HOROWICZet al. 1979a, b; HOLMGRENet al. or the use of alternative metabolic pathways. 1979;ISH-HOROWICZ and PINCHIN1980). All three Use of DEB as a mutagen: At the time that we of the 87C1hsp70 genes can be deleted without initiated our analysis of the 67A-D region, there had greatly affectingviability or fertility (GAUSZet al. 1979; been few reports in the literature about the use of ISH-HOROWICZand PINCHIN 1980). Even whena DEB as amutagen. OLSEN and GREEN (1982) and strain of flies deficient for all but one of the hsp7O SHUKLAand AUERBACH (1980) hadreported the genes at 87A7 was mutagenized, no lethal or visible generation of DEB-induced intergenic deletions on mutations at 87A7 were identified out of over 10,000 the X chromosome, but in general, little was known progenyscreened (GAUSZ et al. 1981). While one about the mechanism of action and efficiency of this mightconclude from these analyses that hsp70 is mutagen in Drosophila. Our use of this mutagen to completely dispensable, it seems more reasonable to generate mutations within the 67A-D subinterval has conclude that hsp68 (which has approximately 75% revealed a number of things. First, the mutational sequence identity with hsp70; HOLMCRENet al. 1979) efficiency is roughlycomparable to that of EMS or one of the three constitutively expressed hsp70 (0.0061 us. 0.0070; see Table 1). Second, with the cognates (INGOLIAand CRAIG1982; CRAIG,INGOLIA, exception of Df(3L)29A6, all of the lethal mutations and MANSEAU1983) provide the same function, and recovered with this mutagenbehave as apparent thuscompensate for the lack of hsp70. Similarly, point mutations in complementation analysis; none mutational analysis of the hsp70gene family of appear to be intergenicdeletions by this criteria. Saccharomyces cereuisiae has shown that disruption of Furthermore, all are cytologically wild type. Thus, if any one of thegenes alone is essentially without any of these mutations are deletions, they are intra- consequence. Only when two or three of the genes genic deletions. Notably, in a recent molecularanalysis are simultaneously disrupted is a mutant phenotype of 21 DEB-induced mutations of the rosy locus, only observed(CRAIG and JACOBSEN 1984;LINDQUIST 2 were found to be intergenic deletions (REARDONet 1986). Likewise, disruption of either of the two yeast al. 1987). Of the other 19, 7 were intragenic deletions genes encoding hsp82 results in no obvious mutant ranging from 50 to 250 base pairs in length and 12 phenotype (FINKELSTEIN,cited in LINDQUIST1986). had intragenic alterations too small to be detected by In light of the above, it is quite likely that mutations the methods used. Third, in contrast to EMS which in any one of the small hsp genes will be covered by has a tendency to induce multiplelesions on the same the remaining functional genes andwill not result in chromosome, DEB appears togenerate predomi- a lethal (or other mutant) phenotype. The fact that nantly single-site mutations. For those complemen- flies which are homozygous for a nonfunctionalallele tation groups with two or more DEB-induced alleles, of hsp28 (hp28"")are viable and fertile bothat normal the mutant homozygotes, in general, have the same growth temperatures and following heat shock (EIS- lethalphase. Moreover, the lethalperiod of the SENBERG and ELGIN1987; LEICHT1987) argues in mutant homozygotes is the same as that of individuals favor of this idea. Another point which supports this which carry the mutation over a deletionof the locus, viewis that our genetic analysis of the67A2-Bl arguing against the presence of accessory lethal mu- subinterval indicates there should be on the order of tations on the chromosome. eight vital loci in this region. Yet, there are seven Genetic analysisof the 67B 1 heat shock geneclus- genes (the foursmall hsp genes and the threerelated ter: On thebasis of tests for (1) heat-inducedlethality, genes) at 67B1 alone, implying that there should be (2) missing or altered proteins on two-dimensional at least 15 essential genes in the67A2-Bl region. protein gels, and (3) restoration of viability by a This difference in predicted number of genetic loci transformant chromosome carrying wild-type copies can be explained if the small hsps are functionally of hsp22, hsp23, and hsp28, none of the six lethal equivalent. Alternatively, this difference may indicate complementation groups in the 67A2-Bl region ap- that mutations of the small hsp genes do not result pear to represent mutations within the small hsp gene in a visible or lethal phenotype as appears to be the 592 B. G. LeichtJ. and J. Bonner case for the single hsp26 gene of S. cerevisiae (PETKO of the 87C region of the third chromosome of Drosophila and LINDQUIST1986). Thus, asanticipated atthe melanogaster. Genetics 93: 917-934. outset of our genetic analysis of the 67 region, it GALSZ, H.J., GYURKOVICS,G. BENCZE, A. A. M. AWAD,J. J.HOLDEN and D. ISH-HOROWICZ,1981 Genetic characterization of the appears that it will be necessary togenerate small region between 86F1,2 and87Bl5 on chromosome 3 of deletions which remove theentire small hspgene Drosophila melanogaster. Genetics 98: 775-789. cluster in order to assess the phenotypic consequences GAGSZ,J., L. M. C. HALL, A. SPIERERand P. SPIERER, of the absence of these genes. 1986 Molecular analysis of the rosy-Ace region of Drosophila melanogaster. Genetics 112: 43-64. Wewish to thank ADELAIDECARPENTER for providing HACKMAN,R. H., and M. GOLDBERG,1968 A study of a melanic Df(3L)AC1, GLENCOLLIER and KATHLEENMATTHEWS for each mutant of the blowfly Luciliacuprina. J. Insect Physiol. 14: supplyinga lethal mutation in the67A2-Bl subinterval, and 765-775. ROBERTCOHEN andROMAN KLEMENZ for providing the transfor- HILLIKER,A. J., S. H. CLARK,A. CHOVNICK andW. M. GELBART, mant stocks used to construct thedouble-transformant stock 1980 Cytogenetic analysis of the chromosomal region im- described herein. We also thank RAY BAYNES forhis assistance mediately adjacent to the rosy locus in Drosophila melanogaster. with the screens over Df(3L)ACI. This work was supported by the Genetics 95: 95-1 10. National Institutes of Health grant GM26693 to J.J.B.; B.G.L. was HOCHMAN,B., 1976 The fourth chromosome of Drosophila melan- supported by a National Institutes of Health Genetics Training ogaster. pp. 903-928. In: The Genetics and Biology of Drosophila, Grant. Vol. Ib, Edited by M. ASHBURNERand E. NOVITSKI.Academic Press, New York. LITERATURECITED HODGETTS,R. B., and A. CHOI,1974 Alanine and cuticle matu- ration in Drosophila. Nature 252: 710-71 1. 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C., E. BERGER,K. SIROTKIN,M. A. YUND,D. OSTERBUR synthesis in imaginal discs of Drosophila melanogaster. Assay by and J. FRISTROM,1982 Ecdysterone induces the transcription in situ hybridization. Chromosoma 58: 87-99. of four heat-shock genes in Drosophila S3 cells and imaginal BONNER, J. J.,C. PARKS,J. PARKER-THORNBURG,M.A. MORTIN discs. Dev. Biol. 93: 498-507. and H. R.B. PELHAM,1984 The use of promoter fusions in ISH-HOROWICZ,D., and S. M. PINCHIN,1980 Genomic organiza- Drosophila genetics: Isolation of mutations affecting the heat tion of the 87A7 and 87C 1 heat-induced loci of D. melanogaster. shock response. Cell 37: 979-991. J. Mol. Biol. 142: 231-245. BRIDGES,P. N., 1941 A revised map of the left limb of the third ISH-HOROWICZ,D., S. M. PINCHIN,J. GAUSZ,H. GYURKOVICS,G. chromosome of Drosophila melanogaster. J. Hered. 32: 299-300. BENCZE,M. GOLDSCHMIDT-CLERMONTandJ. J. HOLDEN, CHENEY,C. M., and A. 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