Genetic Analysis of "Transvection" Effects Involving Contrabithorax Mutations in Drosophila Melanogaster

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1146-1150, February 1988 Genetics Genetic analysis of "transvection" effects involving Contrabithorax mutations in Drosophila melanogaster (transcription/cis control of gene expression/bithorax complex/somatic chromosome pairing) Jost L. MICOL AND ANTONIO GARCIA-BELLIDO Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientfficas, Universidad Aut6noma de Madrid, 28049, Madrid, Spain Contributed by Antonio Garcia-Bellido, October 12, 1987

ABSTRACT Contrabithorax (Cbx) alleles are dominant 17, 18) as if transvection could only occur in the presence of mutations in the Ultrabithorax (Ubx) gene of Drosophia that an active zeste gene. Recently, it has been shown that the cause its ectopic expression in the mesothorax. We have zeste protein binds to a regulatory region of the gene white studied the role of the homologous chromosome in the meso- and to the 5' region of the Ubx transcription unit in the thoracic phenotype in several Cbx beterozygotes. None of the Ultrabithorax (Ubx) gene (19). Cbx alleles studied shows variations in phenotype with extra Genetic studies of the BX-C indicated that the Ubx gene is doses of the Ubx gene. Only Cbx9 and CbxIRM (a revertant of not normally expressed in the mesothorax of wild-type flies Cbx') show synapsis-dependent gene expression ("trans- but it is in flies carrying Contrabithorax (Cbx) alleles (20). vection"). The mesothoracic phenotypes of CbXlRM and Cbx2 This was confirmed by using antibodies against Ubx proteins heterozygotes are strongly modified when the homologous (Ubx-P) in the corresponding imaginal discs (21-23). Thus, chromosome carries breakpoints proximal to or at the Ubx in Cbx/+ heterozygotes the wild-type allele of Ubx is locus or nuli alleles of this gene. These lesions in the homolo- expected to be inactive in the mesothorax. However, Lewis gous chromosome enhance the Cbx9 mutant phenotype and (16) and the present results show that the mesothoracic reduce that of CbXJRM one. The genetic analysis of these phenotype of some Cbx alleles is affected by the genetic transvection effects suggests that the transcription of the nature of the homologous chromosome in heterozygotes. CbXIRM and Cbx2 alleles depends on RNAs of short radius of In this work we analyze the phenotypes of different Cbx action from the homologous Ubx gene. alleles in heterozygotes carrying chromosome breakpoints close to or at the Ubx locus, deficiencies, and point mutations to determine the nature of the putative elements The mutant phenotypes of certain heterozygotes are en- involved in synapsis-dependent expression of the Ubx gene. hanced by chromosomal rearrangements. This phenomenon, The phenotypes of some Cbx alleles when heterozygous called "transvection" by Lewis (1), is thought to be due to a suggest that they are affected by transvection and that this is perturbation of somatic pairing, presumably necessary for possibly mediated by Ubx RNAs of short radius of action. the normal expression of genes. Synapsis-dependent com- plementation was first found modifying the expression of MATERIALS AND METHODS some alleles of the bithorax complex (BX-C), but similar With the exception of CbXIRM, UbXMNI, and UbxMXJ7, all cases have also been observed for the phenotypes of muta- genetic variants and chromosomes used in this work have tions at other loci-e.g., white (2-6), decapentaplegic (7, 8), been described (ref. 24 and other references indicated in Sgs4 (9, 10), and cubitus interruptus (11) of Drosophila. text). CbXIRM (Contrabithorax 1 revertant of Madrid) arose These genetic inferences are supported by cytological obser- spontaneously in a y su-Cbx vf3sa/FM7a;Cbx'e/T(2;3)apxa vations in polytene chromosomes showing puffing depen- stock. UbxmXl7 is a weak, homozygous viable, Ubx allele dent on synapsis of homologues (9, 12) and by mitotic (A. Busturia, personal communication). UbxMN' is an ex- recombination data indicating tight pairing of homologous tremely weak Ubx allele, induced by ethylnitrosourea (J. sequences in interphase nuclei of somatic cells (13). Botas and J.L.M., unpublished). Both are cytologically To account for transvection effects, Ashburner (14) pro- normal. The Za alleles used were Za69-2 and Za69-3. posed that asynapsis would prevent the normal transfer of Variations in expressivity of the mesothoracic phenotype activator signals from an intact regulatory region of one of CbXIRM are defined in five phenotypic classes (see Fig. 2). chromosome to the structural one of its homologue. Three The Cbxi mesothoracic phenotype varies in expressivity and different hypotheses have since been advanced for the penetrance. Penetrance is measured as the percentage of nature of the elements involved in the synapsis-dependent wings showing any transformation to haltere. At least 50 interactions between homologues. (i) Jack and Judd (6, 15) individuals of each genotype were scored, except in some proposed that these interactions are mediated by diffusion of poorly viable genotypes, such as Cbx2/Ubx heterozygotes unstable RNAs produced at the interacting sites. Lewis (16) (minimum of 5). We have used the FP3.38 monoclonal invoked a cis-regulatory entity with a limited effective radius antibody to monitor the expression of Ubx-P in imaginal of action in the nucleus. (ii) Bingham and Zachar (3) sug- discs as described (21-23). gested that synapsed alleles are positioned at specific com- partments of the nucleus, whose local properties are respon- RESULTS sible for transvection effects. (iii) Zachar et al. (5) suggested The CbXlRM Mutation. Cbx)RM arose in a Cbx' chromo- that cis-acting DNA enhancer-like sequences may also trans- some. Cbx' is an x-ray-induced mutation in the Ubx gene activate transcription in synapsed chromosomes. associated with the transposition of a 17-kilobase (kb) frag- Several authors have reported that loci exhibiting trans- ment from the bxd unit of transcription to the 5' region of the vection effects also interact with certain zeste alleles (5, 8, Ubx unit (Fig. 1) (20, 25). Cbx' causes a dominant partial transformation of wing tissue into haltere in the mesothorax. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: BX-C, bithorax complex; Ubx-P, Ultrabithorax pro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. teins. 1146 Downloaded by guest on September 27, 2021 Genetics: Micol and Garcfa-Bellido Proc. Natl. Acad. Sci. USA 85 (1988) 1147

-100 -80 -60 -40 -20 0 20 Kb W- tk.- ...--

r: D

Hm bxdlOO bxdl A

3' Ubx unit 5' 3 bxd unit 5'

.... Er- Ubx922 Ubx195 Ubxl

FIG. 1. Molecular map of the Ubx domain of the BX-C showing B mutations used in this study. Deletions appear as rectangles and insertions appear as triangles. Primary transcripts and exons are represented by heavy lines. Arrows indicate breakpoints. Horizon- tal bars indicate extents of uncertainty in map positions. Ubx'95 is --.S.... I. associated with a single nucleotide substitution (from data in refs. . 25-28). .. vs ( :s~.~ This phenotype is correlated with the ectopic presence of Ubx-P in the corresponding territories of the wing imaginal FIG. 2. Wing phenotypes for genotypes representative of phe- disc (22, 23). Cbx' is homozygous viable and viable in notypic classes of Cbx'RM and Cbx. (A) Class A; Dp(3;J)P15; heterozygotes with Ubx recessive lethal alleles. It has, in CbxIRM/Df(3R)P9; wild-type wing. (B) Class B; CbxIRM/TM1; very weak Cbx transformation, with reduced alula and wing surface, and addition, weak recessive metathoracic and first abdominal spread wings. (C) Class C; CbxIRM/TM6B; weak Cbx transforma- transformation phenotypes, which are detected when it is tion, with alula absent, wing surface moderately reduced, and heterozygous with some lack of function mutations in the spread wings. (D) Class D; Cbx'RM/Dp(3R)P5; moderate Cbx Ubx gene (29, 30). For Cbx' there is only a weak transvec- transformation, slightly weaker than that of class E. (E) Class E; tion effect reported: Cbx'Ubx'/+ + individuals have a very CbxJRM/+; strong Cbx transformation, with posterior compartment slight Cbx transformation, whereas Cbx'Ubx'/R(+ +) flies of wing almost completely transformed into haltere. This latter have wild-type wings (16, 29). The mesothoracic transfor- phenotype is slightly weaker than that observed in Cbx'/+ flies. (F) Example of Cbx2/Jn(3R)Ubx'30 wing-to-haltere transformation mations of Cbx' and Cbx'Ubx' remain unaffected by the (x20). presence of extra doses of the Ubx gene, suggesting that only paired homologues affect the Cbx' expression. breakpoints in the homologue and the degree of CbxJRM Analysis of the CbXIRM mutation shows it to differ from transformation was observed. Interestingly, CbxJRM/Dp- the Cbx' mutation. Like Cbx', the CbXIRM chromosome (3;3)P5 individuals [Dp(3;3)P5 is a small tandem duplication does not show cytologically visible aberrations. It is lethal of the BX-C] show a mesothoracic transformation (class D when or when reces- homozygous heterozygous with Ubx phenotype; see Fig. 2D) weaker than CbXlRM/+, consistent sive lethal alleles. This lethality is due to Ubx insufficiency [it can be rescued with an extra dose of BX-C; Dp(3;1)P115]. Table 1. Variations of the mesothoracic phenotypes of CbXIRM It may result from a second-site Ubx mutation, judging from and Cbx2 depending on the presence of breakpoints in the the metathoracic phenotypes of heterozygotes over reces- homologous chromosome sive mutations of the Ubx gene (see below). This second Mesothoracic mutation may be the cause of the reversion of the Cbx' Cytological positions phenotype. Unlike Cbx', the Cbx phenotypes of CbXJRM Homologous phenotype of breakpoints in the heterozygotes largely depend on the nature of the homolo- chromosome CbXIRM* Cbx2t homologous chromosome: gous chromosome (Fig. 2; see Tables 1 and 2). CbXJRM/+ +§ E 2-11 individuals have a mesothoracic phenotype (Fig. 2E) slightly T(2;3)apXa A/B 70 89C 89E %A weaker than Cbx'/+ flies (class E phenotype; see Fig. 2 for TM] B 29-66 63C 69E 72E 89B 91C 97D degrees of expressivity). That phenotype is further reduced In(3R)8IF; ifthe homologue is structurally aberrant (Fig. 2 B and C) and 88B;91D B 41 81F 88B 91D abolished if the homologue carries a deficiency for the Ubx MKRS B/C 23 71B 87EF 92E 93C gene (Fig. 2A). As was the case for Cbx', the Cbx)RM TM3 B/C 12-24 65E 71C 76C 79E 85E 92D 93A mesothoracic phenotype does not vary with extra copies of 94D 100C 10OF the BX-C in the genome, as duplications in either the X TM6B C 32 61A 63B 72E 75C 84B 84F 86B chromosome (Dp(3;J)P115 and Dp(3;1)05) or the 3L chro- 87B 92D 94A 10OF 10OF mosome arm (Dp(3;3)P146) (data not shown). In(3LR)P30 C 64 64CD 82A Variations in the Dominant, Mesothoracic Phenotype of Jn(3L+3R)P C 22 63C 72E 89C 96A CbxlRM. Whereas the dominant mesothoracic transformation In(3LR)P88 C 66 61A 89CD of CbXJRM/+ is similar in trans heterozygotes with wild-type In(3L)P E 5 63C 72E third chromosomes, the phenotype varies when it is hetero- In(3R)C E 11 92DE 10OF zygous with chromosomes bearing rearrangements (Table Tp(3R)sbd'°4 E 10 1). All balancer chromosomes tested produce phenotypes 91B,89B 89C'91B$ with partial suppression of the mesothoracic phenotype. *Uppercase letters indicate phenotypic classes (see Fig. 2). Trans heterozygotes with inversions affecting only the 3L tValues indicate penetrance (as defined in Materials and Methods). tBX-C is located at 89E; the centromere is at 80. Breakpoints falling arm or regions on the 3R arm distal to BX-C show the same in the critical region are shown in boldface type. phenotypes as do CbXlRM/+ controls. Inversions with at §CbXJRM/+ and Cbx2/+ phenotypes were studied in outcrosses to least one breakpoint proximal to the BX-C (between the Vallecas, Canton S, Oregon R, Gonzdlez Byass, Osborne, complex and centromere) suppress, in a variable extent, the Vifhuelas, and Australia wild-type flies. Cbx transformation. No obvious relation between number of $89B-89C segment is translocated to 91B. Downloaded by guest on September 27, 2021 1148 Genetics: Micol and Garcfa-Bellido Proc. Natl. Acad. Sci. USA 85 (1988) with the known effect of tandem duplications on chromo- not associated with chromosomal rearrangements but carry- some pairing (31). ing small DNA lesions. The latter group includes Ubx' (1, Lewis had shown that breakpoints affecting transvection 25), UbxJ9S (28), and Ubx9_22 (28, 32, 33), whose molecular in the BX-C must fall in a "critical region" between the nature is well known (Fig. 1), and also UbX6JD, UbxMX7, centromere and the complex, suggesting that chromosome and UbxMN', three very weak Ubx alleles, homozygous pairing is initiated at the centromere and proceeds distally viable and cytologically normal (see ref. 34 and Materials (1). Our preceding results indicate that inversion or translo- and Methods). Whenever the CbXlRM/ Ubx combination was cation breakpoints in the critical region, but not deletions, lethal, lethality was rescued by the Dp(3;J)PJ 15, which as are involved in transvection. However, deletions for the Ubx seen above does not affect the Cbx phenotype but rescues gene are as effective reducing the Cbx phenotype as break- lethality and the associated metathoracic lack of function. points within the gene. This indicates that the homologous As shown in Table 2, UbXMN', the weaker allele, slightly chromosome contributes to the transvection effects either by reduces (class D), UbX6JD and UbxMXl7 moderately reduce structural DNA pairing of the Ubx region or by Ubx gene (class C), and the remaining Ubx alleles completely suppress products acting on the paired genes. (class A) the CbXlRM phenotype. These findings favor the To discriminate between these two alternatives we have second hypothesis. studied the trans heterozygotes of CbXJRM and mutations To further define the nature of these diffusible gene falling in different regions of the Ubx gene (Table 2; see also products with effects on transvection, we have studied Fig. 1). We compare the phenotypes of CbXJRM heterozy- recessive mutations in the Ubx gene corresponding to small gotes with Ubx alleles of three different types: (i) deficien- deletions or insertions of transposable elements in introns of cies for the Ubx gene [Df(3R)P9 and Df(3R)Ubx'9, (ii) Ubx the Ubx unit or in the bxd unit of transcription. As seen in alleles associated to breakpoints in the Ubx unit of transcrip- Table 2, in most trans heterozygotes for single recessive tion [In(3R)Ubx'30 and In(3R)Ubx'°], and (iii) Ubx alleles mutations, the mesothoracic transformations were similar to CbXlRM/+ controls. The exceptions are abx', abx2, and bx3. Table 2. Mesothoracic phenotypes of genotypes involving Thus, CbxJRM/abx' and CbxJRM/abi individuals show CbX)RM or Cbx2 and homologous chromosomes carrying weaker Cbx transformations (class C), and CbXJRM/bri flies mutations in the Ubx gene have a class D phenotype. As shown in Fig. 1, the two abx Mesothoracic mutations correspond to deletions and bx3 corresponds to a phenotype gypsy insertion. We have further compared the phenotypic effect of groups of two or more recessive mutations when Genotype CbXIRM Cbi heterozygous with CbXJRM (Table 2). CbxlRM/bxipbxJ indi- + E 2-11 viduals show a moderate reduction of the mesothoracic Dp(3;J)PJ15;+ E 6 transformation (class C) and CbxlRM/bx'bxd' shows a Dp(3;3)PS D 8 slighter reduction (class D). These results suggest that the Dft3R)Ubx'09 Lethal >99 product(s) of the whole Ubx domain, especially those con- Dp(3;J)P15; Dft3R)Ubx'° A 96 taining the abx region, is involved in the interaction between Dft3R)P9 Lethal >99 homologues. Dp(3;J)PJ15; DfJ3R)P9 A 94 Transvection effects are only weak in the metathorax. In(3R)Ubx'30 Lethal >99 CbXJRM fails to complement all abx, bx, pbx, and bxd Dp(3;1)P115; In(3R)Ubx'30 A >99 recessive alleles, the double heterozygotes showing the In(3R)Ubx80 Lethal >99 recessive metathoracic phenotype (data not shown). Con- Dp(3;J)PJ15; In(3R)Ubx80 A >99 trarily, Cbx' does not show mutant metathoracic phenotypes Ubx' Lethal 85 when heterozygous with bx', bx4e, bxd', bxdK, bxdsJJ, and Dp(3;1)PJ15; Ubx' A ND pbxi and shows only weak ones with abx', abx2, bx3, and Ubx'95 Lethal 27 pbx' alleles (ref. 29; unpublished results). All metathoracic Dp(3;1)PJ15; UbxJ9S A ND phenotypes involving CbxIRM are obviously rescued by Ubx9.22 Lethal >99 duplications of the Ubx gene. These results suggest that Dp(3;J)PJ15; Ubx922 A ND CbXJRM has an associated lack of function component stron- Ubx6]D C 10 ger than that of Cbx'. UbXMX]7 C 12 Variations in the Mesothoracic, Dominant Phenotype of UbXMNI D 8 Cbi. Cbxi is another Cbx allele (34) associated with a abx' C 6 breakpoint in the 5' region of the bxd unit of transcription abx2 C 7 (26) [cytologically is In(3R)89E;91C-E]. Its mesothoracic bx3 D 11 phenotype also shows transvection effects in those combi- bx' E 10 nations used in the CbXIRM study (Tables 1 and 2). The Cbi bx34e E 16 phenotype has full penetrance in heterozygotes with defi- bxd' E 31 ciencies and Ubx lethal recessive alleles, the exception being bxd5' D 27 Ubx'95. This penetrance is very low in heterozygotes with bxdK E 42 either normal chromosomes or single recessive mutations, pbx' E 18 other than those mapping in the bxd unit of transcription. pbx2 E 5 The penetrance is intermediate in Cbi heterozygotes with abx'pbx' C 12 chromosomes carrying breakpoints in the critical region or abx'bx3pbx' C 11 mutations affecting the bxd unit, in all cases higher than in bx3pbx' C 20 Cbi/+. Notice that the same genetic combinations have bx'bxd' D 14 opposite effects in CbxJRM and Cbxi heterozygotes, enhanc- Tp(3;3)bxd'°° Lethal 70 ing one and reducing the mutant phenotype of the other. The Dp(3;J)P115; Tp(3;3)bxd'°° A/B ND results of CbX2 heterozygotes suggest that in this case the bx34eDft3R)bxd]21 Lethal >99 homologue causes Cbx2 repression. As in the case of Dp(3;1)PI 5;bxI4eDf(3R)bxdI21 B ND CbxlRM, the phenotype of Cbx2 is not affected by extra Phenotypes are classified as in Table 1. ND, not determined. doses of BX-C. Downloaded by guest on September 27, 2021 Genetics: Micol and Garcia-Bellido Proc. Natl. Acad. Sci. USA 85 (1988) 1149

Interactions of CbXIRM and Cbi with za Alleles. Several the second mutation responsible for the CbXlRM behavior authors (8, 17, 18) have reported that null alleles of zeste (za must be different from the bxd' and bx3 mutations and may alleles) exaggerate the transvection effects of Ubx alleles in be similar, albeit weaker, to Ubx' in its transvection effects. the metathorax. We have studied the effects of two Za alleles In spite of the peculiar interactions with abx alleles, the on the CbXI RM and Cbx2 mesothoracic phenotypes. mutation responsible for the CbxIRM behavior cannot be an CbxJRM/+ males, carrying z' mutations, show a severe, abx allele, because abx alleles do not show bxd- and pbx- class B, reduction of its mesothoracic phenotype. za;Cbx7l+ associated insufficiencies, as does CbxIRM. males show increased penetrance (59%) compared to RNAs Are Involved in Transvection. The mesothoracic z+;Cbxi/+ controls (6%). Thus, as was observed in other phenotypes of CbxIRM and Cbx2 depend on the nature of the cases of transvection of Ubx alleles in the metathorax, Ubx gene in the homologous chromosome but are not CbxIRM and Cbx2 alleles also interact with za alleles in the affected by the presence of extra doses of this gene, trans- mesothorax. located to other places in the genome. This indicates that the Transvection Phenotypes Visualized in Wing Imaginal transvection effects are related to interactions between Cbx Discs. We have monitored the presence of Ubx-P in the wing and its paired Ubx homologue in structurally normal chro- disc in different genotypes showing transvection effects by mosomes and are therefore not mediated by Ubx-Ps. The means of a monoclonal antibody, anti-Ubx-P. There is a pairing-dependent behavior could operate by two alternative complete correspondence between position and extent of the mechanisms. Transvection could be mediated (i) by DNA adult phenotypes (mesothoracic transformations) and posi- structural elements of the Ubx domain-i.e., enhancer-like tion and extent of Ubx-P label in the discs of third-instar elements that act in cis and trans configurations, as in the larvae (results not shown). Zachar et al. (5) model-or (ii) by diffusible gene products Other Cbx Alleles. We have also studied the Contrabitho- with short radius of action-i.e., normal RNA transcripts or rax alleles Cbx3 (26, 34), Hm (26, 34) (Fig. 1), and CbxM' (35) their splicing products, as in the Jack and Judd (6) model. in their interactions with variants in the homologous Ubx Several lines of evidence favor the second alternative. The locus (data not shown). We have not found phenotypic variations of the Cbx's phenotypes by abnormal homologous variations in the mesothoracic phenotypes of their heterozy- Ubx genes could result from the absence of diffusible prod- gotes in the presence of extra doses of BX-C. Nor do the ucts. Six of the Ubx alleles tested, all cytologically normal, phenotypes of heterozygotes vary in an appreciable amount either reduce (UbXMNJ, UbX61D, and UbxMXJ7) or abolish when the homologous chromosome carries breakpoints (Ubx', UbxI9s, and Ubx922) the mesothoracic excess of proximal to the BX-C or lack of function mutations in the function due to CbxIRM and act in opposite ways upon Cbx2 Ubx gene or in the presence of za mutant alleles in the expression (with the exception of Ubx'95). The mutational genotype. origin of the former three Ubx alleles suggests and the Interactions Among Cbx Alleles. Double heterozygotes for molecular nature of the latter three indicates that they a pair of Cbx alleles show additive phenotypes in the correspond to mutations in different exons (see Fig. 1). mesothorax (L. Garcia-Alonso and A.G.-B., unpublished). Transvection in CbxIRM is affected by deletions (abx) and However, CbXIRM/CbX2 individuals show the phenotype also by certain transposon insertions (bx3) in introns but not expected from transvection: a CbxJRM phenotype as in by other transposon insertions. Transvection in Cbxi is only CbXIRM/R(+) heterozygotes (class B) accompanied by Cbx2 affected by certain bxd mutations but not by weak Ubx phenotype with low penetrance as in Cbx2/+ flies. Dp(3;1)- alleles or by deletions or transposon insertions in the Ubx P]J5;CbxlRM/CbxIRM individuals have a very weak (class unit. To explain these results by DNA enhancer sequences B) phenotype. we would have to postulate that these sequences are spread all over the Ubx domain. DISCUSSION The different effects of mutations on CbXJRM and Cbx2 The genetic behavior of CbxlRM and Cbx2 chromosomes is could be explained by the effects of abnormal transcripts. similar to that of previously described examples of transvec- The general effects of Ubx lethal mutations (Ubx', Ubx'95, tion in the BX-C, showing phenotypes dependent on the and Ubx922) could be due to general depletion of Ubx structural nature of the homologous chromosome and being RNAs. The Doc insertion in Ubx' can provide transcription affected by z" mutant alleles. In addition, they show several termination signals responsible for the absence of down- features relevant to the understanding of the transvection stream RNAs. Ubx922 deletes the acceptor for splicing phenomena and to the normal control of Ubx transcription. signal at the end of the abx and bx intron, probably making CbxIRM and Cbx2 alleles show a wide range of phenotypic impossible the separation of the intron from the primary variations in the mesothorax depending on the nature of the transcript (28). But Ubx'95 seems to have only one nucleo- homologue. The same homologues show opposite effects in tide change in the - 50-kb microexon (28). It is difficult to heterozygotes, enhancing the Cbx2 phenotype and reducing imagine in which way this point mutation can affect the the CbXIRM one. The adult phenotypic variations correspond correct splicing (for the same reason it cannot account for an with topographical variations in the distribution of Ubx-Ps in enhancer role of the affected DNA sequence). Why these imaginal discs. Ubx alleles, but not other mutations in the gene, affect CbXlRM Carries a Recessive Lethal Ubx Allele. CbXJRM CbxIRM and Cbx2 remains unexplained. To account for the spontaneously arose in a Cbx' chromosome but both have different behavior of CbxJRM and Cbx2 we would have to mesothoracic phenotypes, suggesting that the original Cbx' propose different roles of Ubx products in the control of mutation, responsible for the abnormal ectopic expression in either CbXIRM or Cbx2. These differences transfer the prob- the mesothorax, remains in CbXIRM. Since CbXIRM behaves lem to the nature of both types of Cbx mutations. The effects as a recessive lethal Ubx allele, with metathoracic and first of abx mutations could be due to the lack of specific RNA abdominal lack of function phenotypes, possibly a second sequences involved in the regulation of CbxIRM. The effects mutation is responsible for the Ubx metathoracic phenotype of bxd mutations could be due to the lack of the correspond- and for the extreme transvection effects found in the meso- ing RNA sequences involved in Cbx2 regulation. thoracic phenotype. The genetic behavior of CbxlRM resem- A Model for the Mechanism of Transvection. We postulate bles that of the combination Cbx' Ubx' (16, 29). Other (i) that the Ubx gene present as duplications in the genome mutations in the Ubx gene also modify in cis the expression does not effectively pair with the Ubx gene in normally of the mesothoracic Cbx' transformation (bx1 and bxd'), but ordered chromosomes, (ii) that Cbx mutations associated none of these phenotypes varies by transvection (36). Thus, with breakpoints (with the exception of Cbx2) similarly Downloaded by guest on September 27, 2021 1150 Genetics: Micol and Garcfa-Bellido Proc. NatL Acad. Sci. USA 85 (1988) prevent pairing with normal homologues, and (iii) that logues would be translated. It is conceivable that the postu- contrary to other Cbx mutations, the structural perturbations lated mechanism of interaction between homologues is at associated with CbXlRM (a 17-kb insertion in the Ubx unit) work in the normal control of expression of the Ubx gene in and with Cbxi (with one breakpoint in the distal 5' region of the metathorax. This is supported by the findings of trans- the bxd unit and the other distal to the BX-C) allow for vection effects for metathoracic phenotypes studied by enough chromosomes in proximity to make possible ex- Lewis (16, 29). That these interactions are mediated by change of RNA products with the homologue. Ineffective RNAs of short radius of action begs us to consider the pairing and therefore undetectable transvection would occur possibility that Ubx trans regulation is mediated by products in the other Cbx mutations (Cbxt, Hm, and CbxMI) associ- of genes that affect the structure and function of these ated with breakpoints in the Ubx domain and in the critical RNAs, in addition to other gene products that may act at the region. We now propose that the CbXlRM chromosome fails transcriptional level. to produce an RNA necessary for its own expression, and Cbxi fails to produce another RNA effective in its own We are grateful to J. Botas, M. P. Capdevila, and J. Castelli-Gair repression. These RNA products, acting in the proximity of for their suggestions and constructive criticism, to R. A. H. White the site in which they are synthesized, would effectively and M. Wilcox for providing FP3.38 antibody, and to E. B. Lewis, diffuse between homologues only when they are paired. G. Morata, and D. C. Otteson for mutant strains. We especially Both types of RNAs are not provided by the homologous thank M. Ashburner for his comments on the manuscript. This work chromosomes carrying Ubx recessive lethal alleles or by was supported by grants from the Comisi6n Asesora de Investiga- deficiency chromosomes and would be partially defective in ci6n Cientffica y Tdcnica and the Fundaci6n Juan March. J.L.M. is chromosomes carrying abx and weak and strong Ubx alleles, a postdoctoral fellow of the Fundaci6n Juan March. in the case of CbxJRM, and would be partially absent in 1. Lewis, E. B. (1954) Am. Nat. 88, 225-239. chromosomes carrying strong Ubx and bxd mutations in the 2. Bingham, P. M. (1980) Genetics 95, 341-353. Cbxi interactions. The absence of these RNAs from the 3. Bingham, P. M. & Zachar, Z. (1985) Cell 40, 819-825. homologue, or the perturbation of pairing by breakpoints in 4. Davison, D., Chapman, C. H., Weeden, C. & Bingham, P. M. (1985) the critical region, results in lesser expression of CbXIRM and Genetics 110, 479-494. higher expression of Cbxl. The role of zeste gene products 5. Zachar, Z., Chapman, C. H. & Bingham, P. M. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 337-346. could be precisely involved in the processing and splicing of 6. Jack, J. W. & Judd, B. H. (1979) Proc. Natl. Acad. Sci. USA 76, these RNAs or in mediating their anchorage to their DNA 1368-1372. target sequences, as postulated in other cases of transvec- 7. Gelbart, W. M. (1982) Proc. NatI. Acad. Sci. USA 79, 2636-2640. tion (5, 8, 17-19). 8. Gelbart, W. M. & Wu, C. F. (1982) Genetics 102, 179-189. 9. Korge, G. (1981) Chromosoma (Berlin) 84, 373-390. This interpretation leads to the following paradox: If both 10. Kornher, J. S. & Brutlag, D. (1986) Cell 44, 879-883. Cbx chromosomes use RNAs from their normal homologues 11. Stem, C. & Kodani, M. (1955) Genetics 40, 343-373. in the mesothorax, both Cbx and homologue chromosomes 12. Ashburner, M. (1%7) Nature (London) 214, 1154-1160. must be transcriptionally active in this segment. The tran- 13. Garcfa-Bellido, A. & Wandosell, F. (1978) Mol. Gen. Genet. 161, scriptional activity of the normal chromosome could be 317-321. 14. Ashburner, M. (1970) Proc. R. Soc. London Ser. B 176, 319-327. constitutive or secondarily appear induced by the constitu- 15. Judd, B. H. (1979) in Eucaryotic Gene Regulation, ICN-UCLA Sym- tive activity of the Cbx chromosome. In situ hybridization posia on Molecular and Cell Biology, eds. Axel, R., Maniatis, T. & Fox, with DNA probes of the Ubx domain to early wild-type C. F. (Academic, New York), Vol. 14, pp. 107-115. embryos (27, 37-39) or imaginal discs (39) has not detected 16. Lewis, E. B. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, RNAs in parasegment 4 or its imaginal 155-164. counterpart, the 17. Kaufman, T. C., Tasaka, S. E. & Suzuki, D. T. (1973) Genetics 75, anterior mesothorax, where some Cbx mutations are ex- 299-321. pressed. Certainly no Ubx-P appear in the same region in 18. Babu, P. & Bhat, S. G. (1981) Mol. Gen. Genet. 183, 400-402. wild-type embryos or imaginal discs (21). This consideration 19. Benson, M. & Pirrotta, V. (1987) EMBO J. 6, 1387-1392. 20. Lewis, E. B. (1978) Nature (London) 276, 565-570. opens the question of the molecular nature of the Cbx 21. White, R. A. H. & Wilcox, M. (1984) Cell 39, 163-171. mutations that cause the transcription of Ubx in these 22. White, R. A. H. & Akam, M. E. (1985) Nature (London) 318, 567-569. regions (22). These Cbx mutations could cause failures in the 23. Cabrera, C. V., Botas, J. & Garcfa-Bellido, A. (1985) Nature (London) mechanism of control of transcription of Ubx by its own 318, 569-571. 24. Lindsley, D. L. & Grell, E. H. (1968) Carnegie Inst. Washington Publ. constitutive products. Alternatively, all of the Cbx mutations 627. would lead to their own derepression and this, in turn, would 25. Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, lead to that of the homologue, by way of RNAs, when P., Lewis, E. B. & Hogness, D. (1983) Science 221, 23-29. effectively paired. 26. Bender, W., Weiffenbach, B., Karch, F. & Peifer, M. (1985) Cold Spring That the homologous Harbor Symp. Quant. Biol. 50, 173-180. chromosome in the mesothorax is 27. Hogness, D. S., Lipshitz, H. D., Beachy, P. A., Peattie, D. A., Saint, not only active transcriptionally to produce RNAs but that R. B., Goldschmidt-Clermont, M., Harte, P. J., Gavis, E. R. & Hel- these are functional ones in translation is inferred by the fand, S. L. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 181-194. following argument. Cbx'/Df(3R)P9 flies have slighter me- 28. Weinzierl, R., Axton, J. M., Ghysen, A. & Akam, M. (1987) Genes Dev. 1, 386-397. sothoracic transformations than Cbx'/+ (30), suggesting 29. Lewis, E. B. (1955) Am. Nat. 89, 73-89. that the Cbx' chromosome does not need the activity of the 30. Casanova, J., Sinchez-Herrero, E. & Morata, G. (1985) J. Embryol. homologue for its transcription but promotes it. CbXJRM/ Exp. Morphol. 90, 179-1%. Df(3R)P9 flies (lethality rescued by BX-C duplications) have 31. Roberts, P. A. & Broderick, D. J. (1982) Genetics 102, 75-89. 32. Kerridge, S. & Morata, G. (1982) J. Embryol. Exp. Morphol. 68, normal mesothoraces. This indicates that the CbX)RM/+ 211-234. mesothoracic mutant phenotype is caused at least in part by 33. Beachy, P. A., Helfand, S. L. & Hogness, D. (1983) Nature (London) expression of the normal homologue, induced by CbXJRM, 313, 545-551. and that the CbXJRM gene is transcribed in the presence of 34. Lewis, E. B. (1982) in Proceedings of the Ninth Congress of the International Society of Developmental Biologists, ed. Burger, M. M. some RNAs of the activated homologue. (Liss, New York), pp. 269-288. The previous discussion indicates that the mutational 35. Botas, J., Cabrera, C. V. & Garcia-Bellido, A. (1988) Development, in nature of the Cbx chromosomes causes their own derepres- press. sion, which, in turn, leads to the derepression of the homo- 36. Lewis, E. B. (1963) Am. Zool. 3, 33-56. logue, and both chromosomes are 37. Akam, M., Martfnez-Arias, A., Weinzierl, R. & Wilde, C. D. (1985) then involved in their Cold Spring Harbor Symp. Quant. Biol. 50, 195-200. mutual transcriptional regulation by way of RNAs of short 38. Akam, M. & Martfnez-Arias, A. (1985) EMBO J. 4, 1689-1700. radius of action. Thereafter, some products of both homo- 39. Akam, M. (1983) EMBO J. 2, 2075-2084. Downloaded by guest on September 27, 2021