Drosophila Pseudoobscura

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Structural Changes in the Antennapedia Complex of Drosophila pseudoobscura

Filippo M. Randazzo,’ Mark A. Seeger,*Catherine A. HUSS,Margaret A. Sweeney, Jeffrey K. Cecil and Thomas C. Kaufman’ Howard Hughes Medical Institute, Institutefor Cellular and Molecular Biology, and Programs in Genetics and Cellular, Molecular and Developmental Biology, Department of Biology, Indiana University, Bloomington, Indiana 47405 Manuscript received October 16, 1992 Accepted for publication January 27, 1993

ABSTRACT The discovery of the striking positional conservation between the Antennapedia and Bithorax homeotic gene complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters has had a substantial impact on our understanding of the evolution of development and its genetic regulation. Structural differences do exist among the mammalian Hox complexes and the ANT-C in D. melanogaster. To gain further insight into the evolutionary changes among these complexes, the ANT-C was cloned in the closely related species, Drosophila pseudoobscura. The overall structure of the ANT-C in D. pseudoobscura is highlysimilar to its D. melanogaster counterpart; however, two differences in the organization of the ANT-C have been identified. First, the 22 gene, a member of the ANT-C in D. melanogaster, is not present in the D. pseudoobscura ANT- C and is possibly absent from the D.pseudoobscura genome. Second, the orientation of the Deformed gene is inverted in D. pseudoobscura, providing it with a 5’to 3‘ direction of transcription identical to the remaining ANT-C homeobox genes with the exception of fushitarazu. These differences demonstrate that subtle changes can occur in ANT-C structure during relatively short periods of evolutionary divergence, although the fundamental organization of the complex is conserved. These - - v observations and others suggest that the complex is not absolutely rigid but that selective pressures have maintained this organization of genes for some Functional reason that remains elusive.

HE discoveries of theAntennapedia Complex PANGand EICHELE 1989; IZPISUA-BELMONTEet al. T (ANT-C) and the Bithorax Complex (BX-C) in 199 l), and the German cockroachBlattella germanica Drosophila melanogaster (KAUFMAN,LEWIS and WAKI- (Ross and TANAKA1988) and the red flour beetle MOTO 1980; LEWIS1978) and the subsequentisolation Triboliumcastaneum (STUARTet al. 1991).Genetic of cognate Hox complexes in mammals(DUBOULE and analysis ofTribolium has uncovered a combined DOLLE 1989;GRAHAM, PAPLOPULUand KRUMLAUF ANT-C/BX-C-like gene complex referred to as the 1989; BONCINELLIet al. 1988) have contributed sig- HOM-C (BEEMANet al. 1989). Its member genes are nificantly to an understandingof the evolution of the functionally homologous and similarly aligned to the developmental process and its genetic control. It has ANT-C and BX-C homeotic genes of Drosophila(BEE- become increasingly clear that not onlyare genes that MAN et al. 1989). Furthermore, four duplicated sets encode metabolically important products conserved of genes with high sequence similarity to theANT-C/ across phyla, but alsoloci that encode developmentally BX-C genes has been found in the mouse (Hox) and importantgene products. Among the develop- in humans (HOX)(see AKAM1989 forreview). These mentally important genes, homeobox genes have been observations taken together indicate that the homeo- isolated from a large array of eucaryotic species (see box must have been present early on in the evolution SCOTT, TAMKUN,and HARTZELL1989 for a review). of eucaryotes,and a combinedANT-C/BX- C-like Perhaps more significantly, clustered sets of homeo- complex must have been present before protostomes box genes have been discovered in humans (BONCI- and deuterostomes diverged over 500 million years NELLI et al. 1988), mice (DUBOULEand DOLLE 1989; ago. GRAHAM,PAPLOPULU and KRUMLAUF 1989;KAPPEN, The structure of the ANT-C and the BX-C in SCHUGHARTand RUDDLE1989), chickens (WEDDEN, D. melanogaster is characterized by the unusual proximity ’ Present address: Mount SinaiHospital Research Institute, 600 University of a significant number of developmentally important Avenue, Toronto, Ontario M5G 1x5, Canada. * Present address: Programin Molecular and Cell Biology, Oklahoma genes and by the collinearity between homeotic gene Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, position on the chromosome and their respective func- Oklahoma 73104. ’Present address: Howard Hughes Medical Institute, Department of tional domains along the anterior-posterioraxis ofthe Biology, Indiana University, Bloomington, Indiana 47405. animal. Thenumerous homeobox-containing genes Genetics 133: 319-330 (May, 1993) 320 F. M. Randazzo et al. of both these complexes presumably arose through a the murine HOX complexes and the human HOX seriesof duplication and divergence events (LEWIS complex. 1978). In contrast to the clustered configuration of To address the questions pertaining to the rigidity these genes, inversion events normally tend to rear- of the ANT-C organization and the unique presence range genesover evolutionary time (STURTEVANT and ofnonhomeotic genes withinits boundaries, the TAN1937; DOBZHANSKYand STURTEVANT 1937). ANT-C in Drosophila pseudoobscura was cloned. Pre- Thus, the extreme proximity of the homeobox genes viousstudies have documented numerous chromo- inferssome form of evolutionary constraint on somalchanges in D.pseudoobscura whencompared change.At least twohypotheses could explain the with D. melanogaster, indicatingthe potential for existence of the homeotic gene complexes: (A) these changes in the ANT-C (STURTEVANTand TAN1937; genescannot function or do notfunction as well DOBZHANSKYand STURTEVANT 1937; STEINEMANN, outside the domain of the complexes, e.g., because of PINSKERand SPERLICH1984). The cloning of ANT- possible chromatinstructure requirements; (B) the C from a secondDrosophilid species, estimated to juxtaposition of the member genes and their complex havediverged approximately 46 millionyears ago cis-regulatoryregions makes separation by random (BEVERLY andWILSON 1984), and the previous clon- genetic processes highly unlikely or even impossible, ing of homologous homeobox gene clusters in two thereby maintaining the ancient configuration. mammalian species (mouse and human), estimated to The uniquecharacteristics of the ANT-C in D. have diverged roughly 68 million years ago (BENTON melanogaster necessitate evolutionary analysis in more 1990), presents an opportunity to explore patternsin closely related species to determine the plasticity of the more recently evolutionary history of homeobox the ANT-C structure. The ANT-C containsfive hom- gene complexes. eotic genes that specify head and thoracic segmental identity, Antennapedia (Antp), Sexcombs reduced (Scr), MATERIALS AND METHODS Deformed (Dfd),proboscipedia (pb)and labial (lab) [see Cloning ANT-C The chromosome walk was performed MAHAFFEYand KAUFMAN 1987or KAUFMAN, SEEGER using techniques described in SCOTTet al. (1983). Both and OLSEN1990 forreview]. The ANT-C differs from lambda EMBL-4 (gift of C. LANGLEY,University of Califor- nia, Davis) and lambda DASH-2 (gift of T. HAZELRIGG, the BX-C [see DUNCAN 1987 and PEIFER,KARCH and Columbia University) D. pseudoobscura genomic libraries BENDER 1987 for review] and from the murine Hox were used. The Ayala strain was used to make the EMBL-4 (DUBOULEand DOLLE 1989;GRAHAM, PAPLOPULU library. All clones except for p191, p195, p474, p475 and and KRUMLAUF 1989;KAPPEN, SCHUCHART and RUD- p488 were obtained from the EMBL-4 library. Prehybridi- zation and hybridization were performed using either 4X DLE et 1989) and humanHOX (BONCINELLI al. 1988) SSC, 5X Dendhardt's solution, 0.1% (w/v) SDS and 0.1% complexes in that it carries some unique homeobox (w/v) carrier calf thymus DNA at 65" (interspecies and genesinvolved in establishingthe embryonic body intraspecies hybridizations) or 5X SSC, 5X Denhardts, 0.1 % plan, namely zen,ftr and bcd. The pair rule genefushi (w/v) SDS and 0.1 % (w/v) calf thymus DNA plus either 30% tarazu (ftz) acts to divide the embryo into segments (w/v) formamide (intraspecies) or 50% (w/v) formamide (interspecies) at 42". Intraspecies hybridizations were fol- (WAKIMOTO,TURNER and KAUFMAN 1984;WEINER, lowed by 0.1X SSC, 0.1 % (w/v) SDS, 0.1% (w/v) sodium SCOTTand KAUFMAN 1984), the maternal effect gene py.rophosphate washes (IX wash at room temperature for 4 bicoid (bcd) encodes a morphogen important in estab- mln followed by 2X washes for 40 min at 65'). The same lishinganterior-posterior polarity (FRIGERIO et al. conditions were used for Southern hybridizations. Probes were made by the nick translation method using Amersham 1986; BERLETHet al. 1988),and the zygotic gene kits (Arlington Heights, Illinois). All lambda genomic clones zerknullt(ren) specifies dorsalembryonic structures recovered in this study were mapped with five restriction (WAKIMOTO,TURNER and KAUFMAN 1984;RUSHLOW enzymes (BurnH1, EcoRI, HindIII, Sal1 and XbaI). Cuticle- et al. 1987). In addition, thez2 gene, which is located like genes were identified by Southern blot hybridization using a pIBI5 1 RX 1.5 (a known cuticle gene) subclones as justproximal to Zen, contains a zen-like homeobox probes (FECHTELet al. 1988; FECHTEL,FRISTROM and FRIS- (RUSHLOWet al. 1987;PULTZ et al. 1988). It is ex- TROM 1989) WITHTHE METHODS AND CONDITIONS AS DE- pressed in a pattern similar to that of zen, but has no SCRIBED ABOVE. detectable genetic function (PULTZ et al. 1988). The Genomic Southern blot hybridizations: D. melanoguster ANT-C also carries several other transcription units Oregon R DNA was a gift of S. Horikami, while D. pseudoobscura DNA was extracted using methods described in that do not contain homeoboxes- amalgam (ama) a SCOTTet al. (1983). Genomic DNA was blotted on Nytran member of the immunoglobulin superfamily (SEEGER, filters (Schlechier and Schuell, Keene, New Hampshire). HAFFLEY and KAUFMAN 1988), a and cluster of eight Prehybridization and hybridization were performed using genes that show sequence similarity to cuticle protein 5x SSC, 5x Denhardt's solution, 250 Pg/ml sonicated and genes (FECHTELet al. 1988;Fechtel, Fristrom and boiled calf thymus DNA, 50 mM Nap04 pH 7.0, 0.1% (w/ V) SDS, and 43% (w/v) deionized formamide at 37" (Mc- FRISTROM1989; PULTZet al. 1988; PULTZ1988). The GINNISet al. 1984). Washing was performed first at room presence of these nonhomeotic genes is one differen- temperature for 5 min (3X) and then at 50" for 15 min (2X) tiating characteristic of the ANT-C from the BX-C, using 2x SSC, 0.1% (w/v) SDS solution (MCGINNISet al. Antennapedia ComplexAntennapedia Evolution 32 1 1984). The probes were made usingthe random primer a’* the 3’ or 5‘ half of z2 and then washed under low- P method (FEINBERGand VOGELSTEIN1983). stringency conditions (data not shown). The 3’ probe consists entirely of open-reading frame sequences lo- RESULTS cated directly downstream of the homeobox, while the5‘ probe contains 300 bp of codingsequence, Cloning the ANT-C from D. pseudoobscura: The including the homeobox, and 140 bp of transcribed, ANT-C in D.pseudoobscura (psANT-C) was cloned to noncodingsequence, as determinedfrom previous determine the extent of evolutionary constraints on structural analysis (RUSHLOWet al. 1987). The 12 3’ the structure of the ANT-C in another Drosophilid probehybridized tothe predicted restriction frag- species. A number of separate regions of the psANT- ments from the D. melanogaster genomic clones and C were cloned by hybridizing a D. pseudoobscura ge- to itself, but not to any DNA within the pb-bcd interval nomic lambda phage librarywith D. melanogaster Antp, of D. pseudoobscura (data not shown). A Southern blot ftz, pb and lab derived probes under low stringency probed with the z2 5’ homeobox-containing fragment hybridization conditions (see MATERIALS AND METH- showed hybridization to the 22-containing restriction ODS).Standard chromosome walking techniques were fragments of D. melanogaster genomic DNA and to used to fill in the gaps between the cloned genomic the 22 5’ positive control. There were no strongly entry points. Overlaps between steps in the walk were hybridized bands in the lanes containing the D. pseu- confirmed by Southern blot cross-hybridization and doobscura genomic phage (p300 and p3 10) from the by restriction site mapping. All lambda genomic in- pb-bcd interval. There were however several weakly serts were mapped using five restriction enzymes- hybridizing bands from these latter phages that rep- EcoRI, BamHI, HindIII, Sal1 and XbaI. A few restric- resent cross-hybridization to zen (data not shown). A tion site polymorphisms among overlapping genomic z2 homolog should hybridize more strongly to a z2 clones were identified,and we suspect that they reflect homeobox-containingprobe than the zen homolog. heterogeneity in the nonisogenic flies used to generate Thus, D. melanogasterz2 probes fail to detect a z2 the genomiclibraries. The relative positions of D. homolog in the pb-bcd interval of D. pseudoobscura. pseudoobscura homologs of the ANT-C coding regions The question remains, however, whetherz2 is pres- were first identified by Southern blot hybridization. ent elsewhere in the pseudoobscura genome. To This was done using D. melanogaster cDNAs and cor- D. address this question, D.pseudoobscura genomic responding genomic probes and low stringency conditions (final wash of 2x SSC, 65”; data not shown). Southern blots were hybridized separately with (A) The identity of restriction fragments similar to theD. the zen cDNA, (B) the 3’ half of z2, and (C) the 5‘ melanogaster geneprobes were then confirmed by half of z2 (all of which were derived from D. melano- sequencing selected D. pseudoobscura subclones. The gaster) andthen washed under very low-stringency DNA sequences werethen compared with existing conditions (Figure 4).The zen cDNA probe, a positive ANT-C gene sequences fromD. melanogaster. control,hybridized to the expected zen-containing Gene order remains conserved for ANT-C in D. restrictionfragments in D. melanogaster-7.2 kb pseudoobscura: The ANT-C in D. pseudoobscura has BamHI,1.2 kb BamHI, 5.9 kb HindIIIand 1.4 kb been cloned from a position downstream of the labial HindIII (Figure 4A;see Figure 3 for restriction map). gene to the Antp homeobox. Except for the cuticle- Consistent with our previous Southern hybridization like genes that were identified by Southern blot ho- and DNA sequence date in D. pseudoobscura, the zen mology alone (data not shown, see MATERIALS AND cDNA probe hybridizes to restriction fragments that METHODS), diagnostic DNA sequences identified conform to the predictedsizes of zen-containing frag- ANT-C gene homologs (Figure 1; SEECERand KAUF- ments in D. pseudoobscura-7.4 kb BamHI, 4.7 kb MAN 1990 forbcd sequence) and their precise location BamHI, 6.0 kb HindIII, 10.4 kb EcoRI and 1.2 kb within the psANT-C and the chromosomal positions EcoRI restriction fragments. Also evident are a num- of their D. melanogaster homologs is summarized in ber of other DNA fragments with weaker homology Figure 2. The results demonstrate that Antp,ftZ, Scr, in both D. melanogaster and D.pseudoobscura (Figure Dfd, ama, bcd,zen, pb, the culticle-like gene cluster, 4A). Due to the low stringency conditions used, hom- and lab are clustered and identically ordered in both eobox cross-hybridization is the most likely explana- Drosophila species. tion for the numerous secondary bands. A 22 homolog is apparently absent from D. pseu- No apparent homology tothe 3‘ portion of D. doobscura: One difference between the two species is melanogaster 22 (22 3’) is detectable in D. pseudoobscura the absence of z2 from the bcd-pb interval in D. pseu- (z2 3’ contains the downstream 47% of the 22 open- doobscura (Figure 3). Southern blots of D. pseudoob- reading frame (Figure 4B)). The z2 3’ probe detects scura genomic clones that encompass the pb-bcd inter- D. melanogaster z2, since the D. melanogaster control val, p300 and p3 10, were firsthybridized with D. lanes display strong hybridization to the predicted 9.6 melanogaster genomic DNA clones containing either kb BamHI fragment. In the D. melanogaster HindIII 322 F. M. Randazzo et al.

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FIGURE 1.-DNA sequence comparisons between D. melanogaster (m.; above) and D.pseudoobscura (p.; below) from selected regions in the D. pseudoobscura chromosome walk. The DNA sequence was aligned using the "bestfit" program of the GCG software package (DEVEREUX, HAEBERLIand SMITHIES1984). All alignments use a gap weight of 5.00 and a gap length weight of 0.30. Vertical bars betwen D. melanogaster and D. pseudoobscura DNA sequence indicate nucleotide identity. Vertical arrows indicate intron/extron junctions. The highly conserved AG sequenceimmediately preceding all 3' splice sitesand the highly conserved GT sequencethat immediately Antennapedia Complex Antennapedia Evolution 323 lane,however, strong hybridization signals are ob- However, in contrast, in D. pseudoobscura the 5’ to 3’ served at 12 kb and at4.7 kb, but not at the predicted orientation of the Dfd gene is in phase with the other 6.7 kb HindIII fragment. This discrepancy is most homeobox-containing genes of the psANT-C (Figure likely due to a strain polymorphism. The absence of 2). It seems that an inversion between ama and Scr detectable z2 3‘ homology in D.pseudoobscura indi- has occurred in one of these two species. This is cates thatthe 3‘ half of z2 has divergedbeyond demonstrated by Southern hybridization data using detectability or that z2 does not exist in D. pseudoob- separate D. melanogaster exon-l/exon-2 and exon-3/ scura. exon-4 probes derived fromDfd (data not shown) and The 5’ z2 probe (z2 5’), which contains a homeo- by DNA sequence from p431 and p475. box, hybridizes weakly toan apparently single ge- In contrast to Dfd, the remaining psANT-C genes nomic locus in D.pseudoobscura (Figure 4C) rather remain oriented as in D. melanogaster. DNA sequence than zen (see below). The origin of this signal at 14 analysis from phage p 15 reveals that D. pseudoobscura kb (BamHI), 5.0 kb (HindIII) and 2.8 kb (EcoRI) is ftz remains in the same 5’ to 3’ orientation relative to unclear (Figure 4C). No comparable band is observed the complex as its homolog in D. melanogaster (Figure when D. pseudoobscura genomic DNA, digested with 1). Additionally, DNA sequencedata demonstrate EcoRI, is probed with the zen cDNA (Figure4A). The thatthe orientations of the remaininggenes in z2 5‘ probe also produces a weak signal at the pre- psANT-C are unchanged with respect to their homo- dicted zen positions in D. pseudoobscura, and this prob- logs in D. melanogaster (Figure 1).Since only Southern ably derives from homeobox cross-hybridization (Fig- blot analysis was performed on the cuticle-like genes ure 4C). Taken together, theabsence of detectable z2 in the psANT-C, their 5’ to 3’ orientations are un- 3’ homology and thepresence of weak z2 5’ homology known. suggests at least two alternative possibilities. The first Intergenic distances and large intronlengths are is that z2 is no longer present in the D. pseudoobscura conserved across species:One defining characteristic genome,but another related gene with detectable of the ANT-C is the proximity of its member genes. homeobox homology to D.melanogaster z2 resides Has the distance between adjacentgenes changed over within thegenome. Alternatively, a 22 homolog is 46 million years of evolution? Measurements of the present outside of the pb-bcd interval of D. pseudoob- distances between selected positions (sequencedre- scura, but has diverged significantly in nearly half of gions are identified in Figure 2) within adjacent genes its codingregion. Given the high degree of DNA demonstrate that there is

+5 5

FIGURE2.-Molecular map of ANT-C in D. melunoguster with corresponding D. pseudoobscuru genomic clones. The molecular map of ANT-C in D. melunoguster, depicted above the coordinate line (Scorn et ul. 1983; SEEGER,HAFFLEY and KAUFMAN1988; DIEDERICHet ul. 1989), is divided into four segments, (a)-(d). The first segment (a) starts at the telomere-proximal end of the complex and the last segment (d) ends after the 3' most exon of Antennupediu. Exons are depicted as shaded boxes. Shown below the coordinates (kb of DNA) are the D. pseudoobscuru genomic clones obtained from the chromosome walk. Hind111 restriction sites found within the genomic clones are depicted as thin vertical bars. The thicker vertical bars at the ends of each clone are lambda phage cloning sites (see MATERIALS AND METHODS for description). Lines connecting the D.melanoguster molecular map to theD. pseudoobscuru clones indicate locations of homology as determined by DNA sequence analysis. The hatched bar located directly below the coordinates in (a) delineate the region of Southern blot homology in D.pseudoobscuru to D. melunoguster (ANT-C cuticle gene-like probes). The bcd and umu positions are from SEECERand KAUFMAN(1990) and SEEGER(~~~~). Antennapedia Complex Antennapedia Evolution 325 and the beetle HOM-C suggests that the structures of these complexes are relatively rigid and incapable of undergoing rearrangements thatsurvive evolutionary selection. The observed inversions of the Dfd gene in D. pseudoobscura and theftz transcription unit in Dro- sophila hydei (relative to D. melanogaster) demonstrate H that subtle rearrangements can occur without com- promising gene function(this study and MAIER,PREISS 9.6 B 3.1 B 128 ?.6 B and POWELL1990). Whether theseobserved inver- 3.8R 3.S R 2.4 R S2R 8.3 R sions indicate the potential for other rearrangements that would split the complex is more difficult to determine.It is quite possible that cis-regulatory sequences are rearranged by these inversions, but these (b) D. pseudoobscunr alterations have little effect due to the ability of enhancer elements to work 5’ or 3’ of a gene. Thus, a translocation with an identical breakpoint could dis- lkb- rupt proper gene regulation and result in lethality. Since the cis-regulatory elementsfor Dfd have not 7.1 R 10.4 R 12R 42R 1.6 H 1.0 H 8.7 H 28H &OH 1.SH been fully defined and the precise extents of these H HH R H HRR HR H inversions are not known, the impact of these inver-

”_ sions on the rigidity of the ANT-C is difficult to assess B a e 8.5 B 6.6 B 4.7 B 7.4 B completely. FIGURE3.-Restriction maps of the pb-zen interval in D. rnelano- Interestingly, the inversion of Dfd and ftz, in D. gaster and D.pseudoobscura. Restriction map of the pb-ren interval pseudoobscura and D. hydei, respectively, likely repre- in (a) D. melanogaster and (b) D. pseudoobscura (SEEGER1989), (B) sents the orientation of these genes in an ancestral BamHI, (H) Hind111 and (R) EcoRI. The stippled boxes represent complex. The homeobox genes of the ANT-C pre- exon-2 of pb, the striped box represents the 22 transcription unit, sumably arose from a series of duplication and diver- the black boxes represent Zen, and the white box represent bcd. Drawn, with arrows, above the restriction maps are theboundaries genceevents that must have occurredbefore the of the genomic library clones from the respective chromosome ancestral split between mammals andarthropods. walks. Note that an artifical EcoRI restriction site lies at each of Consistent with this gene duplication and divergence those boundaries. model, one characteristic of the mouse Hox-2 com- quenced positions within exons that border large in- plex is the identical direction of transcription for all trons were compared (Figure 2). Dramatic changes the homeobox-containing member genes (RUBOCKet are not observed for Scr (1%), Dfd (2%)and lab (7%). al. 1990). In D.melanogaster all the homeobox con- On the other hand, a moderate change is observed taininggenes are transcribed in the same 5’ to 3’ for pb (13%). Clearly, gross changes in intron size and direction except for Dfd andftz (KAUFMAN,SEEGER intergenic spacing have not occurred during the di- and OLSEN1990). After the duplication events that vergence of these two species. gave rise to Dfd andftz, separateinversion events most likely resulted in the reversal of orientation of these two genes somewhere in the D. melanogaster lineage. DISCUSSION Whether there is a Drosophilid species that hasall We report here the cloning of the ANT-C from ANT-C homeobox containing genes with the same Drosophilapseudoobscura. The homologs tothe D. direction of transcription, as is seen in the mouse Hox- melanogaster Antp, ftz,Scr, Dfd, ama, bcd, zen, pb,and 2 complex, remains unknown and would require the lab genes, but not z2 are all present in the D. pseu- analysis of additional species. doobscura complex(psANT-C), in the same linear The nonhomeotic genes of the ANT-C are con- order and similarly spaced alongthe chromosome. served in D. pseudoobscura: A number of nonho- The similar arrangement of ANT-C homeotic genes meotic genes reside within the ANT-C of D. melano- in organisms ranging from D. melanogaster to D. pseu- gaster. These include the homeobox-containing gene doobscura to beetles, mice andhumans suggests a Zen, bcd and ftz, and the nonhomeobox-containing functional importance to this organization of genes genes ama and thecuticle-like gene cluster.The origin that natural selection has maintained. of the homeobox containing genes zen, z2 (see below Asmall chromosomal inversion reveals some for discussion), bcd, and ftz presumably derives from flexibility in ANT-C structure: The extraordinary duplication and divergence eventswithin the ancestral positional conservation of homeotic genes within the complex. It has been postulated that the lab and Abd- murine Hox complex, Drosophila ANT-C and BX-C, B genes are among the original homeobox genes of 326 M. F. Randazzo el al.

FIGURE4.-Genomic Southern hybridizations of D. melanogasterand D. pseduoobscura DNA: searching for 22 homology. Southern blot of restriction enzyme digested genomic D. melanogaster (D. mel.) Oregon and pseudoob- (A) zen cDNA probe (B) zz 3' Probe (c) 22 5' probe R D. 0. mel. 0.pso. 0.mel. 0.pso. D. me/. 0.pso. scura (D.pso) DNA-(B) BamHI. (H) Hindlll, (R) EcoRI. DNA size markers consisted of a 1:l mG-2 mxm mixture of lambda phage DNA digested with Hind111 and withHind111 and EcoRI.Random primed S2P-labeledD. melanogaster (A) Zen pB.2 cDNA (1.2 kb EcoRI), (B) 22 3' (0.262 kb EcoRI/ Sall), and (C) 22 5' (0.445 kb Sall/XbaI) probes were hybridized to identical Southern blots for l=d 4.7- 5"> "1 greater than 36 hr using the low stringency conditions of MCGINNISet al. (1984). Specific activity of the probes: (A) 7.1 X IO8 cpm/pg 2.0--> DNA; (B) 5.2 X 10" cpm/pg DNA and (C) 5.6 X 10" cpm/pg DNA. After hybridization, the blots 1.2" were washed under very low-stringency conditions (2X ssc, 50" (McCrNNlset a[. 1984). Low- stringency hybridization and wash conditions plus excess lambda marker DNA relative to any given genomic DNA sequence resulted in strong signals eminating from some ofthe size markers. Proper DNA transfer to the Nytran filters in the D. pseudoobscura lanes is evident from the equal and evensmearing caused by nonspecific hybridization of probe to the genomic DNA. the ancient complex (CRIBBSet al. 1992). If this hy- pseudoobscura may indicatetheir functional impor- pothesis is correct, then zen, 22, bcd and ftz would tance. The presence of nonhomeobox genes has not represent more recent additions to the ANT-C. been reported in the murine Hox or human HOX All of these homeobox-containing genes (except22 complexes or the beetle HOM-C. If they are truly (see below)) are found in the psANT-C complex. In absent from those complexes, it is likely that ama and addition, bothftz andbcd have been identified in other the cuticle-like genes reside in the ANT-C as a result Diptera: ftz in D. hydei (MAIER et al. 1990) and bcd in of recent insertion events and the insertion of these seven different Drosophilid species (MACDONALD genes in the ANT-C may be another example of the 1990;SEEGER and KAUFMAN 1990)and in Musca disintegration of the homeoticgene complexes in domestica (SOMMERand TAUTZ1991). The question Drosophila relative to other organisms (see MCGINNIS whether any of these geneshas a homolog outsidethe and KRUMLAUF1992). Diptera remains unanswered. Attempts to identify a r2 gene evolution: The absence of 22 from the jzhomolog in the red flour beetle and honeybees psANT-C, its apparent absence from the D.pseudoob- have been to date unsuccessful (STUARTet al. 1991; scura genome, and the lack of a detectable function WALLDORF,FLEIC and GEHRING 1989).It is also the for 22 in D. melanogaster raises some interesting ques- case that clear homologs of these four genes have yet tions concerning the origin and function of the 22 to be identified within the murine Hox and human gene. Because of the high degree of relatedness of Zen HOX complexes (see MCGINNISand KRUMLAUF and 22 with respect to homeoboxsequence (75% 1992). Thus, it is temptingto speculate that these amino acid identity), gene structure (-1 0 10 bp vs. genes represent recent additions to the ANT-C and -1350bp transcription unit with one small intron may be limited to the Diptera. However more exten- located just downstream of the ATG translation start sive analyses of thenondipteran examples will be site), and temporal and spatial regulation, these genes required to demonstrate this point. likely arose from a duplication of an ancestral gene The nonhomeobox containing genes of the ANT- followed by divergence in sequence (RUSHLOWet al. C have been conserved in the psANT-C aswell. Hom- 1987). Since the nonhomeobox sequences of zen and ologs to ama andthe cuticle-like genecluster are 22 exhibit noamino acid sequence similarity, the found in the psANT-C at positions similar to their D. divergence between these two genes has been substan- melanogaster counterparts. Although deficiencies re- tial. moving the cuticle-like gene cluster or ama do not A numberof possibilities could explain the apparent have majorphenotypic consequences (PULTZ 1988; absence of 22 in D. pseudoobscura. The 22 gene could M. A. SEECER,T. C. KAUFMANand C. GOODMAN,in have existed before the divergence of the common preparation), the conservation of these genes in D. ancestor to D. melanogaster and D. pseudoobscura, but A ntennapedia Complex Antennapedia Evolution 321 was thensubsequently lostin the D. pseudoobscura First, important transcriptional regulatory elements lineage.Alternatively, 22 may havearisen as a zen might be shared or interspersed within the complex. duplication after the separation of the D.melanogaster These cis-acting elementscould be traditional en- and D. pseudoobscura lineages. Thus, 22 may never hancersequences that are interspersedamong and have been presentin a D.pseudoobscura ancestor. This used by multiple genes, or they could represent ele- explanation is consistent with the absence of a detect- ments that organize chromatin structure throughout ablefunction for z2 in D. melanogaster. Clearly an the complex (see MIRKOVITCH, MIRAULTand LAEM- analysis of other intermediate Drosophilid species will MLI 1984; GASSERand LAEMMLI 1986;GYURKOVICS be necessary to more fully understand the origin of et al. 1990; KELLUMand SCHEDL 1991). In the murine the z2-zen gene pair. Hox complex, preliminary evidencesuggests that reg- Besides the zen-z2 gene pair, two other examples of ulatory elements may be interspersed between genes, adjacentand highly relatedhomeobox genes have thereby potentially restricting the fixation of inversion been described in Drosophila. The gooseberry (gsb) events (RUBOCKet al. 1990; RENUCCIet al. 1992). In locus consists of two adjacent genes, gooseberry-proxi- D. melanogaster, theftz gene is embedded within Scr mal and gooseberry-distal, which possess highly related regulatory regions (PATTATUCCI,OTTESON and KAUF- homeoboxes andother sequence similarities. Al- MAN 199 I), and there is evidence that abd-A and Abd- though the gsb-p gene product accumulates primarily B of the BX-C share some enhancer-like regions (CEL- in thecentral nervous system andthe gsb-d gene INKER et al. 1990).However, further evidence for product in the epidermis, the gsb genes do have some interspersed regulatory elements is lacking for other overlapping sites of expression. These genes presum- ANT-C and BX-C genes. In fact, the three homeotic ably arose by a duplication and divergence event, as genes of the BX-C can be separated genetically with- did zen and 22, but unlike the zen-22 situation, the gsb out detectablycompromising functions (STRUHL patterns of expression have divergedconsiderably. 1984; TIONG,WHITTLE and GRIBBIN1987; however, The engrailed and invected genes represent another see KARCH et al. 1985for contradictoryevidence), set of adjacent homeobox genesthat arehighly related and there are numerousinversions in the ANT-C that in sequence (COLEMANet al. 1987). Similar to Len and break within one genebut fail to affect other adjacent z2, the functional contribution of each gene in these genes, suggesting that genes of the BX-C and ANT- gene pairs has not been defined. C may not share regulatory elements (ABBOTTand The creation of new genes to fulfill novel functions is an essential process in the evolution of more com- KAUFMAN1986; PULTZet al. 1988; DIEDERICHet al. plicated organisms. The presence of these highly re- 1989). Additionally, pb and Zab null mutations have beenrescued using transgenesthat do notcontain lated and adjacent genes, like zen and 22, most likely represent a phase in the evolutionary development of DNA derived from adjacent functional genes (RAN- new genes and functions. Over time some of these DAZZO, CRIBBSand KAUFMAN199 I ; CHOUINARD and duplicated genes will acquire novel functions and be- KAUFMAN 1991).These data indicatethat inter- come important or essential to the organisms while spersed regulatory elements, if present, are not essen- others will be lost. The zen-z2 genepair offers an tial forproper gene functions,although they may excellent opportunity to study this process. The loca- contribute a fine-tuning role that increases the overall tion of zen and z2 in a defined region, the ANT-C, fitness of the organism. our knowledge abouttheir functions in D. melano- The role of chromatin structure in the regulation gaster, and the changes that have occurred during the of ANT-C or BX-C genes is not well defined. Chro- divergence of D. melanogaster and D. pseudoobscura, matin scaffolding attachment sites (SARS) have been make the analysis of the origin of zen and z2 very experimentally defined for sequence surrounding the accessible and attractive. ftz locus (GASSERand LAEMMLI1986), and consensus Why is theorganization of thehomeotic gene sequences characteristicof SARS have been identified complexes conserved? The most striking feature of in upstream regulatory regions of the labial locus (S. the homeotic gene complexes is the collinearity of CHOUINARD andT. C. KAUFMAN,unpublished obser- gene orderon the chromosomewith their domains of vations). In addition, synapsis dependent regulation expression alongthe anterior-posterior axis of the (transvection effects) has been observed for a number embryo. That this aspect of homeotic gene complex of homeotic genes, including Scr in the ANT-C (PAT- organization has been maintained from Drosophila to TATUCCI and KAUFMAN1991). In certain geneticback- human, with over500 million years of divergence grounds, the homologous chromosomesmust be able between these species, is truiyremarkable. Experi- to pair for proper Scr transcriptional regulation. Re- mental evidence that helps to explain this conserved arrangements that prevent pairing disrupt Scr regu- organization is sparse. Several hypotheses, however, lation. These observations reveal a nonessential re- can be proposed. quirement for transcriptionalregulation that is de- 328 F. M. Randazzo et al. pendent on chromosomeorganization and possibly the complexes are conserved as a consequenceof their chromatin architecture. organizational complexity. Although it is likely that The complexity of regulatory elementssurrounding nonessential butadvantageous regulatory elements the homeotic genesin combination with the proximity are interspered within the homeotic complexes, offer- of these cis-acting sequences to adjacent genes could ing a selective advantage to those who maintain this contribute to the conservtion of the homeotic gene organization, it is also possible that the complexity of complexes. These two factors, complexity and prox- cis-regulatory sequences andthe deleterious conse- imity, would substantially increase the probability that quences to ectopic expression contribute to the ob- a random chromosomalrearrangement would disrupt served rigidity of the complexes as well. Of course, proper gene regulation and thus would be eliminated these evolutionarycomparisons are merely suggestive, from the gene pool. The observation that in D. mela- and the proof of these hypotheses will depend on nogaster Scr regulatory sequences extend minimally to further experimental analysis. the 3’ end and possibly within the Ant@transcription unit, with the ftz locus also embedded in this region, We are grateful to CHARLES LANGLEY andTULLE HAZELRIGG for the D. pseudoobscura genomic libraries. We thank DAVID CRIBBS is an excellent example of this complexity and prox- for the pb exon-2 sequence in D. pseudoobscura sequence, DWAYNE imity (PATTATUCCI,OTTESON and KAUFMAN1991). JOHNSON for performinga number of phage preps, SANDI HORIKAMI Other regions of the ANT-C are not as “dense” (see for the D. melanogaster genomic DNA, KIM FECHTELfor the cuticle Figure 2). For example, the distances from ama to Dfd gene probes and DEE VEROSTKOfor her priceless administrative and Dfd to Scr are seemingly quite extensive, although work. We also thank the dissertation committee of F.M.R., a.k.a. THOMASBLUMENTHAL, MARC MUSKAVITCH, ELIZABETH RAFF and the extent of cis-regulatory sequences for these genes SUSAN STROME,for critical reading of this work. This work was has not been precisely defined.A variation of this supported by a National Institutes of Health Predoctoral Fellowship hypothesis, proposed by RANDAZZO,CRIBBS and (GMO7757) to F.M.R. and M.A.S. and an NIH grant (GM24299) KAUFMAN(1991) as the“prisoner effect,” suggests to T.C.K. T.C.K.is an investigator of the Howard Hughes Medical Institute. that rearrangementevents that disrupt thecomplexes are unlikely due to potential deleterious position-effects on homeotic loci. Ectopic expression of homeotic LITERATURECITED genesoften results in lethality. Unlike the first hy- ABBOTT, M. K., andT. 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