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P Transposon-Induced Dominant Enhancer Mutations of Position-Effect Variegation in Drosophila Melanogaster

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P Transposon-Induced Dominant Enhancer Mutations of Position-Effect Variegation in Drosophila Melanogaster Copyright 0 1993 by the Genetics Society of America P Transposon-Induced Dominant Enhancer Mutationsof Position-Effect Variegation in Drosophila melanogaster Rainer Dorn,* Janos Szidonya?’ Gunter Korge? Madeleine Sehnert,*Helge Taubert,* Essmail Archoukieh,* Bettina Tschiersch,*Henning Morawietz,* Gerold Wustmann,*’* Gyula Hoffmannt and Gunter Reuter*” *Znstitutjiir Genetik, Martin-Luther-Universitat, 0-0-4020HallelS., Germany,+Znstitute of Genetics, Biological Research Center .f the Hungarian Academy ofsciences, H-6701 Szeged, Hungary, and SZnstitut fur Genetik, Freie Universitrit Berlin, 0-1000Berlin 33, Germany Manuscript received May 18, 1992 Accepted for publication October7, 1992 ABSTRACT P transposon induced modifier mutations of position-effect variegation (PEV) were isolated with the help of hybrid dysgenic crosses (7r2 strain) and after transposition of the mutator elements pUChsneory+ and P[IArB]. Enhancer mutations were found with a ten times higher frequency than suppressors. The 19 pUChsneory+- and 15 P[lArB]-induced enhancer mutations canbe used for cloning of genomic sequences at the insertion sites of the mutator elements viaplasmid rescue. Together with a large sampleof X-ray-induced (48) and spontaneous(93) enhancer mutationsa basic genetic analysis of thisgroup of modifier geneswas performed. Onthe basis of complementation and mapping data we estimate the number of enhancer genes at about 30 in the third chromosome and between 50 and 60 for the whole autosome complement.Therefore, enhancer of PEV loci are found in the Drosophila genome as frequently as suppressor genes. Many of the enhancer mutations display paternal effects consistent with the hypothesis that some of these mutations can induce genomic imprinting. First studies on the developmentally regulated gene expression of PEV enhancer genes were performed by &plactosidase staining in P[IArB] induced mutations. N recent years it has become apparent that, at a Cytogenetic studies revealed the existence of both I primary level, gene activity is regulated by re- dominant suppressors and enhancersof PEV (REUTER gional changes in chromatin structure. In order to and WOLFF198 1 ; SINCLAIR,MOTTUS and GRICLIATTI identify genes involved in the regulation of changes 1983; REUTERet al. 1986; SINCLAIR,LLOYD and GRIG- in chromatin structure several groups have employed LIATTI 1989). In thesestudies the total number of the phenomenon of position-effect variegation (PEV) PEV modifier genes in the Drosophila genome was andscreened for dominant modifiermutations of estimated tobe about 100-1 20 (HENIKOFF1979; PEV (REUTER and WOLFF1981 ; SINCLAIR,MOTTUS WUSTMANNet al. 1989). By studyingtheir dosage- and GRIGLIATTI1983; LOCKE,KOTARSKI and TARTOF dependent effects four classes of geneshave been 1988; WUSTMANNet al. 1989). Because in PEV gene distinguished: in one class of genes adeficiency causes inactivation is caused by a change in chromatin con- suppression (haplo-suppressors) and in another group densation (heterochromatinization) the dominantsup- of loci a deficiency displays an enhancereffect (haplo- pressor and enhancer mutations isolated were sup- enhancers). For some genes of both classes a duplica- posed to identify loci that might encode structural or tion was found to result in an opposite triplo-effect regulatorychromatin components. Cloning and se- quencing of several of these genes appearsto confirm (haplo-suppressors with a triplo-enhancer effect and this hypothesis (JAMES and ELGIN 1986; REUTER et al. haplo-enhancers with a triplo-suppressor effect). The 1990). Therefore, further geneticstudies of dominant majority of the modifier loci of both classes do not modifier mutationsof PEV were performed todissect display triplo-effects (REUTER and SZIDONYA1983; and analyze the complex genetic basis of chromatin SZIDONYAand REUTER 1988; LOCKE,KOTARSKI and assembly as well as its functional implication in regu- TARTOF1988; WUSTMANNet al. 1989). These dosage- lation of gene activities or epigenetic developmental dependent effects of PEV modifier genes might reflect programs. opposingchromatin condensation and decondensa- tion processes. ’ Present address: University of Horticulture and Food Industry, Depart- The studies of the PEV modifier genes undertaken ment of Plant Genetics and Selection, Menesi str. 44, H-1118 Budapest, Hungary. to date were concentrated almost exclusively on sup- * Present address: Institut fur Neurobiologie und Hirnforschung, Magde- burg. Germany. P.O. Box 68, Germany. pressors. Three of these loci have been cloned(JAMES ’Corresponding author. and ELCIN 1986; REUTERet al. 1990; K. BAKSA,H. Genetics 133: 279-290 (February, 1992) 280 R. Dorn et al. MORAWIETZ,M. J. AXTON, V. DOMBRADIH. TAUB- REUTER,DORN and HOFFMANN1982; REUTER,HOFFMANN ERT, G. SZABO,I. TOROK,A. UDVARDY,H. GYURKOV- and WOLFF 1983). Therefore, E-var(3)1°' was used to test for new suppressors, and Su-var(2)1°'was used to select ICS, D. M. GLOVER,J. GAUSZand G. REUTER,manu- newly induced enhancers. Enhancer E-var(3)1°' is dominant script submitted). They encode a heterochromatin- to the suppressor effect of an additional Y chromosome, it specific protein, a zinc finger nuclear protein, and a effectively excludes the recovery of false positives from the type I phosphatase, respectively. Sequencing of these suppressor mutation screen which arise from nondisjunction genesappears to confirm the hypothesis that such of the Y chromosome. Mutagenized or hybrid-dys enic w'"''/Y males were genes encode regulatoryor structural components of crossed to wm4'; Sco/T(2;3)a$ + In(2L)Cy,apxn Cy E- chromatin. In addition, the mutationsof another sup- var(3)lo'/TM2 females and the offspring w'"'~;+/T(2;3)apX0 pressor locus, Su-var(2)1, which were found to reduce Cy E-var(3)1°'/+flies were inspected for new suppressor H4 deacetylation and chromatin packaging (DORNet mutations. Enhancer mutations were selected in the w'"''; al. 1986), are butyrate sensitive and display a lethal +/T(2;3)apxa Su-var(2)1°'/+ offspring of a cross ofmutagen- ized w'"'" males or femaleswith wm4'; Cy/T(2;3)apx" Su- interactionto extra Y heterochromatin (REUTER, var(2)1°'/Sb females or males. In addition, strong suppres- DORNand HOFFMANN1982). The mutations of two sors can be identified in siblings with the genotype wm4'; Cy/ other suppressor locishow identical pleiotropic ge- +; Sb/+. netic effects (REUTERet al. 1986). P hybrid-dysgenic males were recovered from a cross (at Suppressor mutations of PEV strongly inhibit het- 18") of w~~~ females to r2 males, a strain containing many P elements (ENGELS1989) and crossed to females, as set out erochromatinization of euchromatic regions in varie- above, to screen for enhancers or suppressors. gating rearrangements as shown by cytological analy- The w'"''/, Icarus-neo/+ males were recovered by cross- sis of salivary glandgiant chromosomes (REUTER, ing a strain carrying the lethal insertion at 27A balanced WERNERand HOFFMANN1982) suggesting that these over Cy0 (STELLERand PIROTTA1986) tow'"4h;+/+ females. loci are involved in chromatin condensation (EISSEN- The F1 progeny was heat shocked (37" for 1 hr) during late embryonic and early first larval instar development. BERG 1989; GRIGLIATTI199 1). Alternatively, the re- Transpositions of the pUChsneory+ element were induced ciprocal class of genes which mutate to enhancers of in heterozygotes with a fl Sb P[ry+ A2-31 (99B) (ROBERT- PEV (haplo-enhancer loci) might encode chromatin SON et al. 1988) or TM3,ry Sb e P[ry+ A2-31 chromosome components involved in decondensation processes and (G. REUTER, G. HOFFMANN,R. DORN,J. GAUSZand H. therefore might also be involved in the control of SAUMWEBER,manuscript submitted). In thecrosses different pUChsneory+ insertions, all without effect on PEV, were gene activation. Although the enhancers of PEV con- used: one insertion in 67C, two independent insertions into stitute a functionally interesting group ofloci they the Cy0 chromosome and three different X chromosomal have not been the subject of any detailed genetic and insertions. New transpositions of the pUChsneory+ element molecular studies. were selected asry+ males in the Cy+Sb+ offsprin from a In order to address this lack of knowledge we un- cross ofryJo6 females withCy0 pUChsneory+/+; ryJo'Sb P[r A2-31 (99B) males or as ry+Sb+ males from a cross of ry 2: dertook a series of large scale experiments to isolate females withX pUChsneory+/Y;ryJo6/TM3,9Sb e P[ry+ A2- enhancers of PEV after X-ray mutagenesis to recover 31 males. Only a single ry+ animal was collected from each both rearrangements andsingle site mutations as well vial. Each and tested for its effect on PEV by crossing with as P elementand modified P elementtransposon w""; Cy/T(2;3)apxa Su-var(2)1°'/Sb females. The wm4'/, +/ induced mutations which would be helpful in further T(2;3)apx"Su-var(2)l0'/ryJo6 male offspring were inspected for enhancer mutations, while their wm4'/, Cy/+; Sb/ryJo6 molecular studies. The modified P elements used were siblings were examined for suppressor mutations. Using a pUChsneory+ or P[lArB] to facilitate cloning of en- backcross of wm4'/, +/T(2;3)apxaSu-var(2)loO'/ry506excep- hancer loci. In addition the P[IArB] element, which tional males show an enhancer effect (mottled instead of contains the lac 2 marker, allowed the first insight suppressed red eye phenotype). The putative enhancer can into the developmental and tissue specific expression be localized on the chromosomes with the help of aneuploid segregants of the upxa translocation
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