Feb 5, 1988 - Annie Rowe' and Michael Akam. Department of Genetics, Downing Street, ... Peifer et al., 1987). The bx region lies within the longest intron of ...
The EMBO Journal vol.7 no.4 pp. 1 107 - 1114, 1988
The structure and expression of
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Annie Rowe' and Michael Akam Department of Genetics, Downing Street, Cambridge CB2 3EH, UK 'Present address: Department of Anatomy and Developmental Biology, University College and Middlesex School of Medicine, The Windeyer Building, Cleveland Street, London WIP 6DB, UK
Communicated by M.Akam
We describe the structure and expression of an exceptional bithorax complex mutation, Cl. This deletion mutation removes major portions of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) protein coding regions, yet retains many of the functions normally executed by these genes. We show that the ends of the Cl deletion map to analogous positions within the Ubx and abd-A transcription units, and that the deletion chromosome gives rise to a set of novel fusion transcripts that may encode hybrid abd-A/Ubx proteins. These fusion products are transcribed from the abd-A promoter, but exhibit a novel spatial pattern of expression that combines aspects of the normal Ubx and abd-A distributions. This pattern provides evidence for the existence of regulatory elements located in the 3' region of the Ubx transcription unit that can act on the abd-A promoter at a distance of at least 20 kb. Key words: bithorax complexlUltrabithorax/abdominal-AI regulation/gene fusion
Introduction The homeotic genes of Drosophila act during development to specify the correct identity of each segment (Lewis, 1978). The diversity of all segments from the mesothorax (T2) to the eighth abdominal segment (A8) appears to be con-
trolled by three principal units within the bithorax complex (Sanchez-Herrero et al., 1985). Each of these three units Ultrabithorax (Ubx), abdominal-A (abd-A) and AbdominalB (Abd-B), encodes a family of proteins that incorporate the same homeobox sequence, yet each serves to specify the different identities of several segments. Ubx, for example, is the major determinant of segment identity in parasegments 5 (T2p/T3a) and 6 (T3p/Ala) (Lewis, 1978; Casanova et al., 1985), whereas abd-A is the major determinant in parasegments 7-10 (Sanchez-Herrero et al., 1985; Tiong et al., 1985). The different developmental fates of parasegments 5 and 6 can largely be attributed to differences in the distribution, the amount, and possibly also the precise variant form of the Ubx protein expressed in these two regions (Akam and Martinez-Arias, 1985; White and Wilcox, 1984, 1985; Beachy et al., 1985). The Ubx region of the bithorax complex contains two functional regions in addition to the Ubx protein coding transcription unit. The bithorax (bx) region is required ©IRL Press Limited, Oxford, England
hybrid homeotic
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for the normal development of parasegment 5 (T2p/Ala), whereas the bithoraxoid (bxd) region is required primarily in parasegment 6 (T3p/Ala) (Lewis, 1978; Casanova et al., 1985). Current evidence suggests that these two regions are not 'genes', but extensive regulatory regions which control the expression of the Ubx transcription unit (for review see Peifer et al., 1987). The bx region lies within the longest intron of the Ubx transcription unit, whereas the bxd region lies 5' to its promoter. The suggestion that Ubx proteins mediate both bx and bxd 'functions' is challenged by the existence of one exceptional mutation, Cl. This mutation, originally isolated and characterized as Ubxcl by G.Struhl (personal communication) deletes the entire 5' protein coding exon of the Ubx transcription unit, and yet it retains certain aspects of Ubx function (see below). We show here that the exceptional phenotype of this mutation can be attributed to a hybrid transcription unit which fuses Ubx to the adjacent abd-A gene of the BX-C. This fusion unit is transcribed, and we believe that it generates hybrid proteins which retain many aspects of normal Ubx and abd-A function. The regulation of this hybrid transcription unit supports the model that the bx region of Ubx is a regulatory element capable of acting on remote promoters.
Results The structure of the Cl deletion chromosome Southern hybridization data show that the Cl chromosome is deleted for 100 kb of DNA within the Ubx and abd-A regions of the BX-C (not shown). To define the end points of this deletion we have cloned the novel DNA fragment generated by the fusion of remaining Ubx and abd-A sequences at either end of the deletion (Figure 1; see Materials and methods for further details). The proximal breakpoint lies within the Ubx transcription unit at -51 kb, close to the location of the second microexon. To determine whether this microexon is deleted, we hybridized a Ubx cDNA clone that contains both microexons to cloned genomic sequences of both wild-type and Cl mutant chromosomes spanning the proximal breakpoint. Sequences encoding the second microexon lie within a 900-bp BamHI -AvaI fragment that remains intact in the C] chromosome. This fragment is separated from the deletion breakpoint by 1300-1800 bp. Thus the Cl deletion removes the Ubx promoter, the major 5' coding region and one of the two microexons, but leaves the second microexon and the 3', homeobox-containing exon intact (Figure 1). abd-A is transcribed in the same direction as Ubx, from sequences 30-60 kb upstream of the Ubx promoter (Karch et al., 1985; F.Karch and W.Bender, personal communication). The structure of the abd-A transcription unit is not fully established, but Karch and Bender have sequenced an abd-A cDNA clone that contains an apparently complete -
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A-Rowe and M.Akam
and the Ubx 3' exons will preserve the reading frame of the Ubx sequences, potentially allowing the expression of an abd-A 5'IUbx 3' fusion protein.
Fig. 1. The structure of the fusion mutation joining the abd-A and Ubx transcription units of the Cl chromosome. (A) The relative locations of the abd-A and Ubx transcription units are shown against the molecular map of the BX-C. Coordinates (in kilobases) are taken from Bender et al. (1983) and Karch et al. (1985). Note that the usual genetic orientation of the map has been reversed to bring the transcription units into conventional 5' -3' orientation. The extent of the transcription units is shown by arrows, with exons shaded. The promoter of the abd-A transcription unit has not been located, and the two short introns that lie within the 5' cluster of abd-A exons are not shown. H marks the location of homeoboxes. Hatched boxes delimit putative regulatory regions defined by the loci of subfunction mutations: abx/bx, anterobithorax/bithorax mutations; bxd, bithoraxoid/postbithorax mutations; iab-2, iab 3/4, infra-abdominal mutations. (B) Expanded restriction maps of cloned fragments spanning the deletion endpoints in wild-type (Canton-S) and Cl chromosomes. Hybridization of the Ubx and abd-A wild-type fragments to restriction digests of the Cl fusion confirms that the regions of homology are as shown by the hatched and stippled shading. The precise location of the breakpoint between the SalI and PstI sites has not been determined, but this 1-kb fragment carries homology to both Ubx and abd-A sequences. Solid bars show Ubx and abd-A exons drawn to scale; dotted lines indicate the uncertainty as to their precise location. Restriction sites: A, AvaI; B, BamHI; R, EcoRI; S, SalIl.
protein coding region (personal communication). Exons of this cDNA cluster at either end of a long (20 kb) intron (Figure 1). The distal break of Cl lies within this large intron, 2-2.5 kb from the last exon of the 5' cluster. Thus Cl deletes the 3' cluster of abd-A exons which encode the homeobox and carboxy-terminal regions of the abd-A protein. Both breakpoints of Cl lie in intron sequences. Transcription from the abd-A promoter may therefore generate a primary transcript containing the complete 5' exons of abd-A fused to the 3' exons of Ubx. The splice donor site terminating the last remaining abd-A exon lies in the same translation frame as those in Ubx (F.Karch, personal communication). Thus splicing between the abd-A 5' exons 1108
The Cl deletion retains some Ubx and abd-A functions A priori, we would expect the Cl mutation to eliminate all Ubx and abd-A functions, for it deletes a major part of the protein coding sequences for both of these genes. The phenotype of embryos lacking all Ubx and abd-A functions has been described (e.g. Df(3L)Ubx'09, Ubx- abd-A-; Lewis, 1978; Morata et al., 1983). The most striking feature of such embryos is that the diversity of segments T3 to T4 is abolished; each of these metameres now resembles T2 (parasegment 4), showing sensory organs (Keilin's organs and ventral pits) and cuticular denticles characteristic of thoracic segments. In addition, the posterior abdominal segments (A5 to A7) are transformed to a hybrid segment type, with thoracic sensory structures (Keilin's organs and ventral pits) but abdominal-type denticles. Embryos homozygous for the deficiency Cl express a unique phenotype quite different from that of the Ubxabd-A- embryo (G.Struhl, personal communication; Rowe, 1987; Casanova et al., 1988) (Figure 2). In these embryos, segments T3 to A4 are not all alike; T3 appears to develop normally; Al is thoracic, with Keilin's organs and ventral pits [we feel that it resembles more closely the normal T2 than T3 (cf. Casanova et al., 1988)]. A2 to A4 all show clear abdominal characteristics. Keilin's organs are partially (A2) or completely (A3, 4) suppressed, and the denticle morphology becomes progressively more like that of the normal A2/A4 type in each more posterior segment. Ventral pits remain, however, on all abdominal segments from Al to A7. The denticle belts of A5 to A8 resemble those in a leaky abd-A mutant. The ability of the Cl chromosome to elicit 'abdominal type' development is dosage sensitive. The denticles of segments A2 to A7 all appear more 'thoracic' when only one dose of Cl is present (in the hemizygote) than they do in the homozygote. Similarly, in the hemizygote, the suppression of Keilin's organs is incomplete in A3 and A4
(Figure 2). We conclude from these observations that the Cl chromosome retains some functions that mimic those of the normal Ubx and abd-A genes, and that these functions are expressed differentially in segments T3 to A4. In the accompanying paper, Casanova et al. (1988) show that Cl complements almost completely the abx and bx functions of Ubx, expressed in parasegment 5, but fails to complement the pbx and bxd functions, expressed in parasegment 6. When in trans with iab-2 and abd-A mutations it resembles a leaky abd-A allele. These recessive phenotypes indicate that the Cl deletion reduces normal Ubx and abd-A function, but dominant phenotypes of the Cl chromosome suggest that Ubx and abd-A functions are also inappropriately expressed. Animals of the genotype Cl l + have a near normal adult phenotype. The halteres are slightly enlarged (as we would expect for a Ubx- heterozygote). However, in addition the first abdominal segment of the adult is partially and patchily transformed to a more posterior abdominal character (Rowe, 1987; Casanova et al., 1988). Such weak 'Ultra-abdominal'
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Fig. 2. The ventral surface of cuticle preparations from Cl homozygotes and Cl hemizygotes (CJ/Df(3R)P9). Arrowheads show Keilin's organs, arrows show ventral pits. Denticle belts of each segment are labelled. T2 -3, thoracic segments; Al -8, abdominal segments.
phenotypes have been interpreted as being due to the ectopic expression of abd-A in Al, where this gene would not normally be expressed (Lewis, 1978). Casanova et al. (1988) also report that the Cl chromosome may display a dominant Contrabithorax phenotype. Such transformations were rarely observed in our stocks. Transcription from the C1 chromosome The phenotype of Cl suggests that this chromosome encodes both Ubx and abd-A functions. We therefore looked for any transcripts arising from the remains of the Ubx and abd-A transcription units on the Cl chromosome which could be responsible for these functions. Initially these experiments were carried out by hybridizing Ubx and abd-A probes to size-fractionated embryonic RNA. Sufficient quantities of Cl homozygotes cannot easily be obtained, and so embryos were collected from the progeny of Cl/balancer (= TMI,BX-C+) heterozygotes. This population includes three BX-C genotypes, Cl/C], C1/+ and + / +, all of which survive to late embryonic stages. Half of the RNA in such a population should therefore derive from the Cl chromosome. For convenience we refer to it as Cl/TM] RNA. We have used three probes, Ubx 5', Ubx 3' and abd-A 5' sequences. The patterns of hybridization of Ubx 5' and 3' probes to RNA from wild-type embryos have been described previously (Akam and Martinez-Arias, 1985; Hogness et al., 1985). Both detect the same major transcript species of 3.2
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Fig. 3. Sequential hybridization with abd-A 5' (left) and Ubx 3' (right) probes to a filter carrying RNA extracted from CJITMI embryos. Approximate sizes are indicated for transcripts found in wild-type (+/+) embryos. Novel transcript species in Cl embryos are marked with arrowheads. Complete removal of the abd-A probe after denaturation was checked by autoradiography prior to hybridization with the Ubx probe. Sizes for the abd-A and fusion transcripts have been estimated by comparison with the normal Ubx transcripts. The Ubx 3' probe used here spans the protein coding region of the 3' exon, which is common to the Ubx 3.2- and 4.3-kb transcripts. We have also used a probe specific for the 3' untranslated extension of the 4.3-kb Ubx transcript (subclone A139, Akam and Martinez-Arias, 1985). This probe detects the novel 4.8-kb transcript, but not the 3.2- or 3.5-kb transcripts. 0-6. 6-12, 12-18 indicate age (in hours) of embryos at collection.
and 4.3 kb (Figure 3). These transcripts differ primarily in the extent of the 3' untranslated region. Our abd-A probe is a genomic fragment spanning the second exon of the abd-A cDNA (F.Karch and W.Bender, personal communication). In RNA from wild-type embryos, this probe hybridizes to major transcript species of -5.4 and 4.8 kb (Figure 3). The abd-A 5' and Ubx 3' probes derive from regions which are retained on the Cl chromosome, but the Ubx 5' probe derives from sequences within the deleted region. In Cl/TMI RNA this Ubx 5' probe reveals only transcripts corresponding in size to those present in normal embryos (data not shown). The abd-A 5' and Ubx 3' probes both detect transcripts in the CJ/TMI RNA that are not found in wild-type RNA. These include species of 3.5 and 3.2 kb which hybridize to the abd-A probe, and species of 3.5 and 4.8 kb which hybridize to the Ubx 3' probe. Probing the same filter sequentially with the abd-A 5' and Ubx 3' probe reveals that the normal transcripts of 3.5 kb detected with the abd-A probe comigrate precisely with those detected with the Ubx 3' probe. The 3.2-kb transcript detected with the abd-A probe almost comigrates with the normal 3.2-kb Ubx transcript, making it difficult to determine whether this novel species hybridizes to the Ubx probe. Similarly the 4.8-kb transcript detected with the Ubx 3' probe comigrates with a normal abd-A transcript. These results suggest that at least the 3.5-kb transcript, and possibly all three of the novel transcripts in Cl may derive from transcription and splicing across the fused abd-AI Ubx transcription units. It remains possible that novel tran-
1109
A.Rowe and M.Akam
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Fig. 4. Transcription of abd-A and Ubx in Cl embryos: the extended germ band. (a) Cl homozygote at extended germ band stage hybridized with the abd-A 5' probe. Arrowheads here and in subsequent panels delimit parasegment 6. Transcripts are detected in the ectoderm of parasegments 5-7, and in both ectoderm and mesoderm of parasegments 8-13. Labelling of the amnioserosa (as) is barely detectable above background levels. Most of the hybridization observed with this 5' probe lies over nuclei. (b) Darkfield picture of the same section. (c and d) Adjacent section of the same embryo hybridized with the Ubx 5' probe. There is no specific label. (e and f) Adjacent section of the same embryo hybridized with the Ubx 3' probe. The pattern of labelling is similar to that observed in (a), except that signals are stronger, and a much larger fraction of the label is observed over cytoplasm [cf. Akam and Martinez-Arias (1985) for a similar comparison of Ubx 5' and 3' labelling in normal embryos]. The amnioserosa (as) is clearly labelled.
script species of similar sizes are induced independently in the truncated Ubx and abd-A transcription units, but we consider this to be unlikely. The results presented below show that the spatial and temporal distribution of these novel transcripts appears to be identical, whether they are detected with abd-A 5' or Ubx 3' probes. The pattern of abd-A/Ubx transcription in Cl embryos By using the three probes described above to probe serial sections, we can unambiguously identify the pattern of Ubx and abd-A transcription in embryos homozygous for the Cl deletion. Such embryos show no hybridization with the Ubx 5' probe, and reveal a novel pattern of transcription with both the abd-A 5' and Ubx 3' probes (Figures 4 and 5). This confirms that both Ubx and abd-A transcription units are active in the Cl deletion mutant, despite deletion of the Ubx promoter. In normal embryos, these two probes reveal the very different, but overlapping, distributions of Ubx and abd-A transcripts. In Cl homozygotes, however, we have been unable to detect any difference in the distribution of transcripts revealed by these two probes.
1110
The distribution of Ubx transcripts in wild-type embryos has been described in detail (Akam and Martinez-Arias, 1985). They accumulate from parasegment 5 to parasegment 13, with particularly high levels in parasegment 6. The corresponding distribution of abd-A transcripts spans parasegments 7-13; this is described in more detail elsewhere (Rowe, 1987). When the abd-A probe is hybridized to Cl embryos, transcripts are detected from parasegments 5-13. Thus the abd-A promoter appears to be active more anteriorly than usual, from an antero-posterior level appropriate to the normal expression of Ubx. This altered expression is clearly apparent in the extended germ band, and again in the central nervous system after germ band shortening (Figures 4 and 5). The Cl transcript distribution detected with the abd-A probe differs from that in the wild-type in several other respects. The distribution of transcripts within each parasegment is abnormal, and the levels of C] transcripts in parasegment 13 are comparable with those observed in parasegment 7-12, whereas in normal embryos transcript levels in parasegment 13 are much reduced.
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In all of these respects the distribution of transcripts observed with the Ubx 3' probe mirrors that of the abd-A 5' probe, as we would expect for fusion transcripts. This distribution differs from the normal distribution of Ubx transcripts in several ways: in Cl embryos, parasegment 6 shows low rather than high levels of Ubx expression; transcripts in the ectoderm of parasegment 5 are both more abundant and more uniformly distributed than in normal embryos; transcripts are observed in the visceral mesoderm of the posterior abdominal segments, where abd-A but not Ubx would normally be expressed. Cl transcription in imaginal discs Genetic analysis suggests that the Cl chromosome provides some Ubx functions during adult development-the bx functions are largely retained, although bxd functions are not. We would therefore expect to see transcription from the Cl chromosome in the imaginal discs of the third thoracic segment, which are the precursors for the adult cuticular
type larvae, Ubx is expressed at high levels in the haltere and third leg discs, but abd-A is not expressed in any of the thoracic imaginal discs. (It is presumably expressed in the imaginal histoblasts of the abdominal segments, but these contain very few cells during the larval stage, and we have not been able to detect them in sectioned material.) In the Cli + heterozygotes, abd-A probes hybridize to transcripts in the haltere and third leg imaginal disc. We assume that these are transcripts from the Cl chromosome, but cannot rule out the possibility that the expression of abd-A from the wild-type chromosome may be altered in these individuals. The abd-A homologous transcripts are present at high levels in the anterior regions of the haltere and third leg discs, but at much lower levels, if at all, in the posterior regions of these discs (Figure 6). We do not detect abd-A transcripts in the wing disc, or in any of the more anterior discs of Cli + heterozygous larvae.
structures.
Discussion
Cl homozygotes do not survive long enough to grow imaginal discs, so our analysis has been limited to Cl/balancer heterozygous individuals. These grow to the third instar larval stage with apparently normal imaginal discs. In wild-
The Cl fusion mutation allows us to examine certain aspects of the role and regulation of the Ubx and abd-A genes. However, to interpret the activity of this chromosome, we must attempt to discriminate between abnormal function of 111 1
A.Rowe and M.Akam
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Fig. 6. Section from a Cl/TMI heterozygous larva hybridized with the abd-A 5' probe. W, wing disc; H, haltere disc; L3, third leg disc. Note that parts of the third leg and haltere discs are labelled with the abd-A probe. Examination of serial sections shows that the labelled region lies in the presumptive anterior region of each disc (Rowe, 1987), but we cannot say from these data whether it coincides precisely with the anterior compartment. Scale = 20 /m.
the encoded proteins and abnormalities in their pattern of
expression. Spatial control of transcription Using the assay of in situ hybridization, we have monitored effects on the transcriptional activity of abd-A and Ubx, independent of any question of protein function. We find clear evidence that the deletion of 3' sequences from the abd-A transcription unit and their replacement by 3' sequences from the Ubx transcription unit results in a dramatic change in the spatial distribution of transcripts from the abd-A promoter. We assume that this is an effect on transcription and not message turnover, for there is no evidence that the abd-A promoter is ever active in parasegments 5 and 6 of the normal embryo. We have considered two possible explanations for this effect. Sequences located in the deleted region of the abd-A transcription unit may normally repress its activity in parasegments 5 and 6; alternatively, sequences in the 3' half of the Ubx transcription unit may positively regulate the abd-A promoter in this same region when brought into their new
position.
If the first of these two models were correct, then we would expect other breaks at the same position in the abd-A transcription unit to show similar, adventitious transcription. No available mutant allows us to test this possibility precisely (the most suitably located chromosome rearrangement, abd-AS2, is now extinct). We have however examined transcription from the abd-A promoter in the mutant abd-AMI, which carries a chromosome break at 42 ±fi 1 kb, in the middle of the abd-A transcription unit. We do not see ex1112
pression of abd-A in parasegments 5 or 6 in the embryos homozygous for this chromosome (A.Rowe and E.SanchezHerrero, unpublished results). The model that Ubx sequences are regulating the abd-A promoter of the Cl chromosome receives support from the precise pattern of Cl transcription in parasegments 5 and 6. This resembles the pattern of transcription seen from a Ubx promoter when the bxd regulatory region is deleted, but the bx region remains. Notably, the anterior boundary of expression is indistinguishable to that observed for Ubx; expression is observed in the ectoderm of parasegment 5 but not in the overlying mesoderm; and, in imaginal discs, transcripts are observed in the anterior but not the posterior regions of the T3 discs. We believe that this pattern argues strongly for the existence of positively acting bx regulatory elements that mediate the activation of Ubx in parasegment 5 of the embryo, and in T3a of the developing adult. The implications are that in normal development these sequences act on the Ubx promoter at a distance of > 20 kb, and that this action is largely independent of the effects of the 5' regulatory sequences located within the bxd region. This interpretation is consistent with other genetic analyses of the Ubx domain. Identified bx mutations all map within the region of the Ubx transcription unit that remains intact in the Cl chromosome (Peifer and Bender, 1986). Origin and structure of the products encoded by the C1 chromosome In the accompanying paper, Casanova et al. (1988) show that the ability of the Cl chromosome to express either Ubx or abd-A functions depends absolutely on the integrity of the Ubx 3' exon containing the homeobox. When a deletion mutation in this exon is recombined onto the Cl deficiency, both Ubx and abd-A functions are inactivated, and the phenotype of the Cl chromosome now resembles that of a Ubxabd-A- double mutant. Therefore we are confident that the abd-A functions of the Cl chromosome are mediated by fusion proteins incorporating the Ubx homeobox. We suspect that most or all of the Ubx functions expressed by this chromosome are also mediated by the same family of fusion proteins. The similar distributions of transcripts homologous to the abd-A 5' and Ubx 3' exons argues that the majority of Ubx transcripts observed in Cl homozygotes derive from the abd-A promoter. Moreover, with the sole exception of Cl, all breaks within the Ubx transcription unit inactivate both bx and bxd functions of the Ubx unit, implying that the remaining activity of Cl depends on the associated abd-A sequences. The extent of the Cl deletion is such that the predicted abd-AlUbx fusion proteins will have a sequence organization analogous to that of the normal abd-A and Ubx proteins. The amino terminus of the protein will be entirely abd-A; the homeobox and carboxyl terminus entirely Ubx. The fusion occurs between two sequences that are recognizably conserved in both Ubx and abd-A. One of these is a short conserved peptide found in most Antennapedia-class homeotic genes, the YPWM peptide (Mavilio et al., 1986; Wilde and Akam, 1987); the other is the homeobox (Gehring, 1987). In Ubx, these two motifs are variably separated by 8-51 amino acids, depending on the splicing pathway followed (O'Connor et al., 1988); in the extant abd-A cDNA, they are separated by a sequence encoding 25 amino acids
An abdominal-A - Ultrabithorax fusion gene
(F.Karch, personal communication), and in the predicted fusion proteins, by 24-41 amino acids, again depending on whether or not the remaining Ubx microexon is used. In view of this fortuitous location of the deletion endpoints with respect to coding sequences, it is not surprising that the fusion proteins retain some function. Developmental specificity of the abd-A/Ubx fusion products The unique phenotype of specific segments depends on the normal expression of the set of homeotic genes. According to the classic selector gene model, cells in a single segment would all express the same, unique developmental code, and this code, together with positional information, would direct each cell to follow the appropriate segment-specific developmental pathway (Garcia-Bellido, 1975). It is now clear that the homeotic genes are not uniformly expressed, but are differentially expressed in a complex spatial and temporal pattern within each segment (Akam and Martinez-Arias, 1985; Beachy et al., 1985; White and Wilcox, 1985; Peifer and Bender, 1987). This raises the possibility that the different developmental effects of different homeotic genes result as much from differences in the control of their expression as they do from intrinsic differences in the properties of their products. The phenotype of the Cl fusion provides some support for this possibility. For example, embryos that contain only the Cl fusion gene can express denticle belt morphologies in T3 to A4 ranging from something very similar to the normal T3 to something rather like the normal A4. This must be achieved with a qualitatively reduced spectrum of proteins, and the final phenotype appears to correlate with the level at which the fusion transcript is transcribed in different segments. It is possible that this range of denticle belt morphologies is achieved by the modulated expression of a single
protein species. The phenotype of the Cl mutant suggests that the Cl chromosome is capable of encoding products with bx functions of Ubx, but incapable of encoding products with bxd functions. However, our results are entirely consistent with a model in which it is the regulation of Cl expression that accounts for the phenotype of the mutant combinations, and not the differential activity of the protein products. Thus we have evidence that the Cl fusion is generally expressed at lower levels in parasegment 6 than in parasegment 5, and that this effect is particuarly pronounced in the T3 discs. This would be sufficient to account for the complementation of bx but not bxd/pbx alleles. We hesitate to draw conclusions from our results concerning the relative contribution of the homeobox domain and the amino-terminal domain to the biological specificity of homeotic proteins. To do this, we would need to know that the normal Ubx protein could mediate a particular developmental pathway, but that the abd-A protein could not. We will not be able to assess this question until we know precisely which cells express each of these two proteins in normal development.
Grell, 1968), was used for all our work. The Ubx and BX-C deficiencies, (Ubx- abd-A-),Df(3L)P9 (BX-C-), were obtained from E.B.Lewis. All flies were reared at 25C on yeast-glucose-agar medium.
Df(3L)UBx'09
Cuticle preparations Eggs for embryonic cuticle preparations were collected overnight and aged a further 24 h at 25°C. Unhatched embryos and first instar larvae were mounted as described by Van der Meer (1977). DNA analysis Genomic DNA was prepared from adult flies (Chia et al., 1985) of the stocks Cl/TMI, st pP e1 and from numerous other stocks carrying the TMJ balancer. Southern hybridization (Maniatis et al., 1982) with whole lambda and plasmid subclones of the BX-C DNA (Bender et al., 1983) revealed restriction polymorphisms in the st pP e" chromosome with respect to TMJ and to the standard Canton-S map, but no major structural anomaly in the region -70 to +90 kb. Restriction fragments of the Cl chromosome match those of the st pP e1 l chromosome in the regions -50 to -70, and +50 to +90 kb, but fragments diagnostic for the marker chromosome are missing from DNA of ClITMJ heterozygotes at co-ordinates -45, -20, 0 and +40 kb. Probes from the wild-type BX-C covering the two regions shown in Figure 1 both hybridize to a 9-kb EcoRI fragment that is diagnostic for the Cl chromosome. This putative fusion fragment was cloned by ligating a total EcoRI digest of CJ/TMI DNA into the lambda vector EMBL4 (Frischauf et al., 1983), and screening for phage that hybridized to both proximal and distal breakpoint sequences. Two such phage were obtained, both carrying the diagnostic 9-kb EcoRI fragment. For further characterization a 3.0-kb BamHI-SalI fragment spanning the fusion point was subcloned into the plasmid vector pEMBL8 (Dente et al., 1983). To detect the location of the Ubx microexon, the cDNA clone aDM3602 (Beachy et al., 1985) was nick translated and hybridized to AvaI, PstI and BamHI digests of the fragments shown in Figure 1. Filter hybridization to RNA RNA was extracted from embryos of the indicated ages as described by Henikoff (1983). Total RNA (8 jug) was loaded on each gel track. Formaldehyde gels were run according to Maniatis et al. (1982) and transferred to Hybond-N membrane (Amersham). 32P-Labelled M 13 single-stranded probes were prepared essentially as described by Burke (1984) and hybridized to the filters in 5 x SSPE, 50% formamide at 43°C for 12-24 h. Filters were washed in 0.1 x SSPE, 0.1 % SDS at 60°C. The Ubx 5' template was subclone A128, containing a genomic HindIHl-EcoRI fragment covering the Ubx promoter and the first 400 bases of the Ubx 5' exon. The Ubx 3' template was subclone A134 (see Akam and Martinez-Arias, 1985). The abd-A 5' template was subclone AR153, containing the genomic 1-kb BamHI fragment (52-53 kb) that encodes exon 2 of the abd-A cDNA. In situ hybridization 35S-Labelled M 13 single-stranded probes were prepared and hybridized (in 10% dextran sulphate) as described by Akam and Martinez-Arias (1985). Embryos were fixed and stripped of their vitelline membranes (Akam and Martinez-Arias, 1985), dehydrated and embededded in paraffin wax. Third instar larvae were fixed, dehydrated and embedded in wax as described by Ingham (1985). Sections (6 Arm) were cut, dried onto poly-lysine coated slides, and pretreated as described by Martinez-Arias (1986).
Acknowledgements We thank Gary Struhl for providing us with the Cl mutation, and for drawing our attention to its intriguing phenotype. Francois Karch and Welcome Bender kindly provided unpublished details of the abd-A cDNA clone, while Gines Morata and his colleagues shared with us many of their observations prior to publication. Vivian Irish, Peter Lawrence, Gines Morata and Emesto Sanchez-Herrero provided valuable criticism of the manuscript. This work was supported by the Medical Research Council of Great Britain.
References
Materials and methods
Akam,M. and Martinez-Arias,A. (1985) EMBO J., 4. 1689-1700. Beachy,P.A., Helfand,S. and Hogness,D.S. (1985) Nature, 313, 545-551.
Genetic analysis The mutation UbxCI was recovered by G.Struhl after ethyl methane sulphonate mutagenesis of a chromosome carrying the markers st pP e1l. A stock carrying this marked C/ chromosome, balanced over TMI (Lindsley and
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Received on January 4, 1988; revised on February 5, 1988
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