Copyright 0 1990 by the Genetics Society of America Genetic Analysis of Transvection Effects Involving &Regulatory Elements of the Drosophila Ultrabithorax Gene Jos&Luis Micol,”’ JamesE. Castelli-Gair and Antonio Garcia-Bellido Centro de Biologia Molecular, C.S.I.C.-Universidad Autbnoma de Madrid, 28049 Madrid, Spain Received January 20, 1990 Accepted for publication June 8,1990 ABSTRACT The Ultrabithorax (Ubx) gene of Drosophila melanogaster contains two functionally distinguishable regions: the protein-coding Ubx transcription unit and, upstream of it, the transcribed but non- protein-coding bxd region. Numerous recessive, partial loss-of-function mutations which appear to be regulatory mutations map within the bxd region and within the introns of the Ubx transcription unit. In addition, mutations within the Ubx unit exons are known and most of these behave as null alleles. Ubx’ is one such allele. We have confirmed that, although the Ubx’ allele does not produce detectable Ubx proteins (UBX), it does retain other genetic functions detectable by their effects on theexpression of a paired, homologous Ubx allele, i.e., by transvection. We have extended previous analyses made by E. B. Lewis by mapping the critical elements of the Ubx gene which participate in transvection effects. Our results show that the Ubx’ allele retains wild-type functions whose effectiveness can be reduced (1) by additional cis mutations in the bxd region or in introns of the Ubx transcription unit, aswell as (2) by rearrangements disturbing pairing between homologous Ubx genes. Our results suggest that those remnant functions in Ubx’ are able to modulate the activity of the allele located in the homologous chromosome. We discuss the normal cis regulatory role of these functions involved in trans interactions between homologous Ubx genes, as well as the implications of our results for the current models on transvection. HE Ultrabithorax (Ubx)gene is one of the selector pattern of proteins encoded by the Ubx unit, causing T genescontrolling segmental identities in the absence of UBX in specific regions of embryonic and body of Drosophilamelanogaster. Mutations in this larval tissues (INGHAM 1985; WHITE and WILCOX gene dramatically alter the cuticular structures of the 1985; CABRERA,BOTAS and GARC~A-BELLIDO1985; second thoracic (T2),the third thoracic (T3) and the BOTAS,CABRERA and GARC~A-BELLIDO1988). Muta- first abdominal (Al) segments of the adult body of tions affecting introns of the Ubx unit (abx and bx the fly: gain-of-function alleles of the Ubx gene trans- partial loss-of-function alleles) also cause local abnor- form T2 toward T3 (reviewed in GONZALEZ-GAITAN, malities in the UBX pattern (BENDERet al. 1983; MICOLand GARC~A-BELLIDO1990); loss-of-function PEIFERand BENDER1986; WHITEand WILCOX1985; alleles of the Ubx gene give rise tothe homeotic CABRERA,BOTAS and GARC~A-BELLIDO1985; BOTAS, transformation of T3 toward T2 and/or that of an- CABRERAand GARC~A-BELLIDO1988). terior A1 toward anterior T3 (reviewed in MORATA, Two different classes of apparently null alleles SANCHEZ-HERREROand CASANOVA1986; DUNCAN are known in the Ultrabithorax gene: While abx, bx, 1987; PEIFER,KARCH and BENDER1987; SANCHEZ- pbx and bxd recessive alleles inactivate subfunctions of HERREROet al. 1988; MAHAFFEYand KAUFMAN the Ubx gene, Ubx lack-of-function alleles inactivate 1988). The Ubx gene is composed of three transcrip- tion units (see Figure 1): theUbx unit, coding for most, perhaps all, functions of the gene. Ubx lack-of- mRNAs that are translated into proteins (UBX) with function alleles as a rule cause the absence of func- morphogenetic functions, and the overlapping early tional proteins in the entire realm of action of the bxd and late bxd units coding for RNAs of unknown geneand lethality in homozygous embryos (LEWIS function (BEACHY,HELFAND and HOGNESS1985; 1978; HAYES, SATOand DENELL‘1984). Ubx lack-of- HOGNESSet al. 1985; LIPSHITZ,PEATTIE and HOGNESS function alleles include chromosome rearrangements 1987; O’CONNORet al. 1988). However, mutations in that interrupt the Ubx transcription unit and pseu- the bxd region (bxd and pbx partial loss-of-function dopoint mutations affecting Ubx unit exons (called alleles; BENDERet al. 1983, 1985) affect the spatial pseudopoint as they are not visible in polytene chro- mosomes but usually correspond to mutational dam- ’ Present address: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91 125. age greater thana single nucleotide change) (BENDER ’ To whom correspondence should be addressed. et al. 1983; HOGNESSet al. 1985). Genetics 126 365-373 (October, 1990) 366 J. L. Micol, J. E. Castelli-Gair and A. Garcia-Bellido -120 -100 -80 -60 40 -20 0 20 kb bx” PV td3-” -8xa bxd late untt Ubx unlt bxd early unit 4 1 1s.t n- a/\l/u cONA El0 252L n/ cONA 3601 35 n C cONA 3600 m cONA 3603 FIGURE 1 .-Molecular map of the Ultrabithorax gene showing mutations used in this work. Rectangles indicate deletions and triangles insertions, having the latter the corresponding transposon name (1, 412, harvey, Hobo and Doc). Dotted triangles are gypsy insertions, the arrow indicating when known the direction of their transcription. bx” is associated with two adjacent insertions (gypsy and Doc), the second being irrelevant for tnutant phenotype. ab^".",^ deletion appeared with a Hobo insertion. Df(3R)PY deletes the entire bithorax complex, its left end appearing in the drawing. Df(jR)UbxlUY,which does not appearin the figure, deletes the entireUbx gene and a part of the adjacent abdA gene, its ends would be out of the drawing. Transcription units are indicated as heavy horizontal arrows. Mature RNAs appear under the transcription units, showing exons as rectangles. Capital letters S and L in Ubx transcription unit mature RNAs indicate short and long, corresponding to the use of either of two alternative splicing donor sites in the 5’ exon. All data for this figure were obtained from BENDER et al. 1983, 1985; PEIFERand BENDER1986; LIPSHITZ, PEATTIEand HOGNESS1988; O’CONNORet al. 1988; WEINZIERLet al. 1987. Ubx pseudopoint mutant alleles are not true null to T3 toward T2 homeotic transformations which are alleles: Ubx pseudopoint mutant alleles show in homo- less extreme than those observed for the same reces- zygotes the same phenotypes as null alleles (break- sives over a deficiency for the gene (MORATAand points and deficiencies) (LEWIS 1963; MORATAand KERRIDGE1980; KERRIDGEand MORATA1982; WEIN- GARC~A-BELLIDO1976; KERRIDGEand MORATA1982; ZIERL et al. 1987). This behavior could reflect residual MIGANAand GARC~A-BELLIDO1982; HAYES,SATO and activities of some putative protein productof the Ubx‘ DENELL1984). However, Ubx pseudopointmutant allele. However, several observations strongly suggest alleles are not completely amorphic because their that theUbx’ allele does not produce protein products. trans heterozygotes with recessive alleles have adult First, Ubx‘ homozygous phenotypes are the same as mutant phenotypes less extreme than those observed those observed for breakpoints and deficiencies for for those recessives in heterozygotes with deficiencies thegene in mitotic recombination clones in adults or breakpoints in the Ubx gene (MORATAand KER- (MORATAand GARC~A-BELLIDO1976; KERRIDGEand RIDGE 1980; KERRIDGEand MORATA1982; WEIN- MORATA1982; MIGANA and GARC~A-BELLIDO1982), ZIERL et al. 1987). This inference is supported by in gynanders (LEWIS 1963) and in lethal embryos observations indicating that Ubx pseudopoint alleles (LEWIS1963, 1978; HAYES,SATO and DENELL1984). behave differently than breakpoints and deficiencies, Second, Ubx’ homozygous embryos lack any protein with respect to sensitivity to ether-induced bithorax recognizable by FP3.38 (WEINZIERLet al. 1987), an phenocopies and tointeractions with mutations in the antibody raised against the 5’ exon common to all the Regulator of bithorax gene (CAPDEVILAand GARC~A- mRNAs from the Ubx transcription unit (WHITEand BELLIDO1974, 1978, 1981; J. E. CASTELLI-GAIRand WILCOX1984). Similarly, FP3.38 antibody does not A. GARC~A-BELLIDO,manuscript in preparation). recognize any protein arising from Ubx‘ in larval im- Ubx’ is a pseudopoint Ubx allele lacking detectable aginal discs (CASTELLI-GAIR,MICOL and GARC~A-BEL- protein products: One such pseudopoint Ubx allele is LIDO 1990). Third, the phenotypes of heterozygotes Ubx’ which is associated with the insertion of a Doc involving recessive alleles and Ubx‘ are indistinguish- transposable element in the untranslated region of the able from those involving other pseudopoint Ubx al- 5’ exon of the Ubx unit (Figure 1) (WEINZIERLet al. leles which are known to produce abnormal UBX. 1987). It is not known whether or not this mutation Pioneer observations of LEWIS(1 95 1) (see results perturbs the transcription of the Ubxunit and/or marked with a c in Table 1) showed that some heter- those of the bxd units. Previous genetic studies and ozygotes involving cis double mutants bx Ubx’ give this work show that Ubx’ mutation in trans-heterozy- stronger mutant phenotypes than those involving the gotes with recessive alleles of the Ubx gene give rise single mutant Ubx’. These observations were then C ontrol ElementsUbx Control in Transvection 367 interpreted under the supposition that bx and Ubx TABLE 1 mutations affect two different genes, but cannow be Metanotum and haltere mutant phenotypes of heterozygotes interpreted as suggesting the presenceof remnant involving the U6x' allele functions in Ubx'. The behavior of Ubx'