Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences Are Involved in ...

20 Pages·1998·0.81 MB·English

Copyright(cid:211) 1998bytheGeneticsSocietyofAmerica
Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences
Are Involved in Anchoring Distant cis-Regulatory Regions to the Promoter
La´szlo´ Sipos,* Jo´zsef Miha´ly,*,† Franc¸ois Karch,† Paul Schedl,‡ Ja´nos Gausz*
and Henrik Gyurkovics*
*HungarianAcademyofSciences,BiologicalResearchCenter,InstituteofGenetics,H-6701Szeged,Hungary,†UniversityofGeneva,Department
ofZoologyand AnimalBiology,CH-1211Geneva4,Switzerlandand ‡DepartmentofBiology,PrincetonUniversity,Princeton,NewJersey08544
ManuscriptreceivedJune12, 1997
Acceptedfor publicationMarch 2, 1998
ABSTRACT
The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is
requiredfor the properidentityof the fifth throughtheeighth abdominal segments (corresponding to
parasegments 10–14) of the fruitfly. The morphological difference between these four segments is due
to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-
regulatoryregionsinfra-abdominal-5(iab-5),-6,-7and-8.Thedominantgain-of-functionmutationFrontab-
dominal-7 (Fab-7)removesa boundary separating twoof thesecis-regulatory regions,iab-6and iab-7.Asa
consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in
PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in
Fab-7 flies.Herewe reportthat pointmutationsof theAbd-B gene in transsuppressthe Fab-7phenotype
in a pairing-dependent manner and thus represent a type of transvection. We show that the observed
suppressionis theresultof trans-regulationof thedefectiveAbd-B gene bytheectopically activatediab-7.
Unlikepreviously demonstratedcases of trans-regulation in theAbd-B locus, trans-suppression ofFab-7 is
sensitivetoheterozygosityforchromosomalrearrangementsthatdisturbhomologouspairingatthenearby
Ubx locus. However, in contrast to Ubx, thetransvection weobserved in the Abd-B locusis insensitive to
theallelicstatusofzeste.AnalysisofdifferentdeletionallelesofAbd-Bthatenhancetrans-regulationsuggests
that an extensive upstream region, different from the sequences required for transcription initiation,
mediatesinteractionsbetweentheiabcis-regulatoryregionsandtheproximalAbd-Bpromoter.Moreover,
wefind thatthe amountof DNAdeletedin theupstreamregionisroughlyproportionalto thestrength
oftrans-interaction,suggesting that thisregionconsistsof numerousdiscrete elementsthatcooperatein
tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the
regulation ofAbd-B are discussed.Inaddition,wepresent evidencethat thetenacityof trans-interactions
in theAbd-B gene mayvary, depending uponthe tissue andstageof development.
IN eukaryotes, gene activity can be controlled by ex- such long-distance regulatory interactions is provided
tensiveregulatoryregionsthatarelocatedmanykilo- by the phenomenon of transvection in Drosophila.
bases away from the promoter. In the most widely ac- Transvection, first described by Lewis (1954) in the
cepted model for such long-distance interactions, the Ultrabithorax (Ubx) locus, refers to a partial interallelic
intervening sequences between the regulatory regions complementationthatdependsuponthepairingofho-
andthepromoterarethoughtto“loopout”(Pirrotta mologouschromosomes.Inthebest-documentedcases,
1991). Although the looping model can account for such as in the yellow (Geyer et al. 1990) and the Ubx
long-distance interactionsbetweenregulatoryelements loci (Martinez-Laborda et al. 1992), transvection ap-
andpromoters,italsoposesaproblem.Mostregulatory pears to involve trans-regulation, that is, a regulatory
elements are rather promiscuous in their interactions element on one of the homologues controls the pro-
andarecapableofcontrollingtheactivityofmanydiffer- moteractivityof thecorrespondinggeneontheother,
ent promoters, irrespective of their origin. Thus, the paired homologue. This unusual trans-interaction can
looping model raises the question of how enhancers be exploited to learn more about the mechanisms re-
are able to distinguish their target promoter from the sponsible for specifying conventional interactions be-
promoters of other nearby genes. tween regulatory elements and promoters in cis.
TheUbxgeneisapartofthehomeoticbithoraxcom-
Onemodelsystemforstudying thefactorsgoverning
plex (BX-C). Although BX-C contains only two other
homeotic genes, abdominal-A (abd-A) and Abdominal-B
(Abd-B) (Sa´nchez-Herrero et al. 1985; Tiong et al.
Corresponding author: Henrik Gyurkovics, Hungarian Academy of 1985), these three genes assign proper identity to the
Sciences, BiologicalResearchCenter,Institute ofGenetics, P.O.B.
third thoracic (T3) and all of the abdominal (A1–A9)
521, H-6701Szeged,Hungary.
E-mail:[email protected] segments, corresponding to parasegments (PS) 5 to
Genetics149:1031–1050(June,1998)
1032 L.Siposet al.
PS14. This is achieved through the control exerted by lectedfrom17-hregglaysweredechorionated,fixedanddevi-
tellinized according to the procedure of Mitchison and
PS-specific cis-regulatory elements on the individual
Sedat(1983),modified asdescribedin Karchetal. (1990).
genes of the complex. Thus, abx/bx and pbx/bxd ele-
We modified this procedure further asfollows. The time of
mentsregulateUbxexpressioninPS5andPS6(Beachy fixationwasreduced to12 min. Devitellinizedembryos were
et al. 1985; Hogness et al. 1985; White and Wilcox washedandrehydratedin1(cid:51)PBT(1(cid:51)PBT(cid:53)1(cid:51)phosphate-
1985), and iab-2, -3 and -4 elements regulate abd-A in buffered saline (cid:49) 0.1% Triton X-100 (cid:49) 0.1 bovine serum
PS7,PS8andPS9,respectively(Karchetal.1990;Mac- albumin,1(cid:51) phosphate-bufferedsaline (cid:53)137 mmNaCl,2.7
ias et al. 1990). iab-5 - iab-8 elements regulate the Abd-B mm KCl, 10.1 mm Na2HPO4, 1.8 mm KH2PO4; pH (cid:53) 7.5).
Embryoswereincubatedwithprimaryantibody(monoclonal
class A transcription unit that corresponds to the Abd-B mouse-anti-ABD-B1A2E9;Celnikeretal.1990)dilutedin1(cid:51)
mfunction(Casanovaetal.1986)inPS10throughPS13 PBT 1:1 on a rotating wheelovernight at 4(cid:56)C.Antibody was
(Celniker et al. 1990; Boulet et al. 1991; Sa´nchez- removed, and embryos were washed six times with PBT for
Herrero1991).Specificregulatoryelementsthatregu- about20minonrotatingwheel.Horseradishperoxidase-con-
late the longer transcription units (class B, C and (cid:103); jluugteadteidn rparbebchiti-lalnedti-(m4o(cid:56))uPseBTse1co:2n0d0a,rwyasanadtidbeoddyan(dDAinKcOub)a,teddi-
Zavortnik and Sakonju 1989), corresponding to the withtheembryosonarotatingwheelfor31/2hrat4(cid:56).These
Abd-B r subfunction (Casanova et al. 1986), have not conditions reduced background staining without significant
yetbeenidentified,buttheyareexpectedtospecifythe loss of specificstaining.Embryos were thenwashed againas
expressionpattern oftheclass B, Cand(cid:103)RNAspecies described above. After removing PBT, embryos were briefly
rinsedtwiceinstainingbuffer(0.1mcitricacid, 0.05mNH-
in PS14 andPS15. 4
acetate,pH(cid:53) 5.7adjustedwith NHOH)and stainedin the
Recently,anunusualtypeoftrans-regulationhasbeen mixture of 980 (cid:109)l buffer, 20 (cid:109)l DA4B solution (25 mg/ml
describedfortheAbd-BclassAtranscriptionunitandits stocksolution,finalcc0.5mg/ml)and2(cid:109)lHO (30%stock
regulatory regions (Hendrickson and Sakonju 1995; solution,finalcc0.06%).Thereactionwasstop2ped2 bydilution
Hopmannetal.1995).Incontrasttotheclassicaltrans- with PBT and embryos were rinsed six times with PBT. Em-
vection of Lewis (1955), this trans-regulation is ex- bryoswere stored in PBT with 0.05% NaN3 and mounted in
9:1 glycerol:10 (cid:51) PBT. For the dissection of CNS, we used
tremelyresistant to disruption of homologouspairing.
tungstenneedles.
Inthisarticle,weprovideevidencethattrans-interaction Chromosomes generated by recombination: McpB116iab-7Sz
intheAbd-Bgenemayvaryinitsresistancetodisruption Abd-BD16:The synthesisof this chromosome wasdone in three
of homologous pairing in different tissues. With the consecutive steps.First, we generated the chromosome McpB116
helpofadominantgain-of-functionmutation,Fab-7,we iab-7Szbyrecombination. Out of (cid:122)29,000male progenyof the
crossbetweenMcpB116/iab-7SzvirginsandOregon-Rmales,weiden-
show that transvection of a more regular type, closely
tifiedtwoflieswithdarklypigmentedA4[duetothedominant
resembling theclassical case in the Ubxlocus, can also
mutation,Miscadastralpigmentation(Mcp)]andanadditionalru-
be demonstrated in Abd-B in the adult stage. Detailed dimentaryseventhtergite(iab-7(cid:50)).Inthesecondstep,Microcepha-
analysis of this transvection suggeststhe existence of a lus(Mc) (adominantmutationresultingin astrongreduction
multicomponenttetheringmechanismthatmayensure of the head capsule) was recombined onto the McpB116 iab-7Sz
chromosome.McpB116iab-7Sz/Fab-7McvirginswerematedwithOre-
cis-autonomyoftheAbd-BdomainwithintheBX-C.This
gon-Rmales.Among(cid:122)35,000F males,wefoundthreerecombi-
mechanism involves an extensive region upstream of 1
nantsshowingMcp,iab-7andMc,butnoFab-7phenotype.Finally,
the Abd-B gene, which appears to be distinct from the weisolatedrecombinantsbetweenthechromosomesMcpB116iab-
sequences required for transcription initiation. 7SzMcandAbd-BD16.(McpandMcservedasflankingmarkersto
detectrecombinationevents.)Dp(3;1)bxd111/(cid:49);Abd-BD16/McpB116
iab-7SzMcvirginswereallowedtomatewithOregon-Rmalesand
theirmale progeny((cid:122)90,000) were scoredforthepresenceof
MATERIALS AND METHODS
Mcp and the absence of Mc phenotype. Of the 39 such males
General procedures: Fly stocks were maintained on stan- found, eight were sterile and 23 did not carry Abd-BD16 [based
dardyeast-cornmeal medium.Crosseswereperformedat25(cid:56) oncomplementationwithDf(3R)P9].Theremainingeightlines
enmasse,unlessotherwisenoted.Unlessdescribedinthisarti- carryingtheAbd-BD16weretestedfortheirabilitytosuppressthe
cle, all genetic variants used are described in the following Fab-7 phenotype. Although all of them showed suppression,
references:iab-7MX2,Abd-BD14,Abd-BD16,Df(3R)C4(Karchetal. theyfell intotwodiscrete categories:fiveofthem hadanA6
1985);Abd-BRD18,Df(3R)U110 (Hopmannetal.1995);Abd-BS1, tergite larger than the thin A7(similar to the phenotype of
Abd-BS4(Tiongetal.1985);Abd-BR41,Fab-7,In(3R)Fab-7iab-7R7, Fab-7/Abd-BD16),whereasintheremainingthreethesetergites
Df(3R)R59(Gyurkovicsetal.1990); iab-7164(Celniker etal. wereequallylargebothin A6and inA7. Weconcluded that
1990);iab-7Sz(Gallonietal.1993);McpB116(Karchetal.1994); thefirstgroupcarriedMcpB116only,andthesecondgrouphad
TM3SbP(ry(cid:49))(cid:68)2-3(Reuteretal.1993);UC21-10,1-d(McCall bothMcpB116 andiab-7Sz.Weconfirmed thepresence of both
etal.1994); Cbx1,Df(3R)P9,Dp(3;3)P5,In(3LR)TM1, In(3LR)- lesionsbySouthernblot analysis.The sameprotocolwasfol-
TM3 Sb Ser, In(3LR)TM6B Tb, In(3LR)TM6C Sb, Mc, Tp(3;1) lowed in the generation of recombinant chromosomes that
bxd111,Ubx1, z1,za,zop,zv77h(Lindsley andZimm 1992). In(3R) containedAbd-BD14 instead of Abd-BD16.
Fab-7iab-7R5was isolatedas an X-ray-induced revertantof Fab-7, Fab-7 Abd-BD16: Two apparently contradictory observations
anditsbreakpointwithintheBX-Cwasclonedanddetermined promptedustostudytransvectionintheAbd-Bdomain.First,
bySouthernanalysis.Cytologicalanalysisofpolytenechromo- we found that (when the homologues are paired) an Abd-B
somes was performed as described by Ashburner (1989). point mutation trans to the Fab-7 mutation suppresses the
AdultabdominalcuticlesweremountedasdescribedbyDun- Fab-7 gain-of-function phenotype so that the A6 tergite in
can (1982), and Southern blot analyses were done as de- males is larger than the A7 tergite (trans-suppression, de-
scribedbyBenderetal. (1983). scribedindetailintheresultssection).Second,wenoticed
Immunohistochemical staining of embryos: Embryos col- in an earlier study (Gyurkovics et al. 1990) that when the
Transvection inthe Abd-B Domain 1033
Fab-7 mutationispresentbothin cisandin transtoanAbd-B totheFab-7mutation.]Fab-7UC21-10,1-d/(cid:49)fliesshowamoder-
mutation (Fab-7/Fab-7 Abd-BR41) the A6 and A7 tergites are ateFab-7phenotypethatisstronglyreducedifthesechromo-
equally thin. We presumed that this phenotypic difference somes are over an Abd-B null mutation, suggesting that the
between the two types of mutant combinations was not due Fab-7 phenotype originates mainly from an interaction with
tosome unusualpropertiesofAbd-BR41(whichisanAbd-Bm(cid:50) the paired wild-type Abd-B gene in trans. However, the re-
mutation,Gyurkovicsetal.1990),buttothepresenceofthe maining weak Fab-7 phenotype indicates that UC21-10,1-d is
Fab-7mutationin cistoAbd-BR41.Totestifthis isthecase,we aleakyAbd-Bmutation.Impreciseexcisionsoftheinsertthat
attempted to isolate recombinants carrying both Fab-7 and result in the deletion of the promoter region of the Abd-B
Abd-BD16 (an Abd-B mr null mutation;Boulet etal. 1991)on classAtranscriptionunitshouldeliminatetheremainingFab-
the samechromosome,basedontheassumptionthat Fab-7/ 7phenotypeinfliesheterozygouswiththenullmutationAbd-
Fab-7Abd-BD16 fliesshouldhave a phenotypesimilarto Fab-7/ BD16.(The excision of theP element can be detectedby the
Fab-7 Abd-BR41. For this purpose, ry506Fab-7/Abd-BD16 females lossof thery(cid:49)markergene carriedbythe UC21-10,1-dinser-
werecrossedtory506Fab-7/ry506Fab-7males,andthemaleprog- tion.) We therefore isolated nine ry(cid:50) mutations with no re-
eny were scoredfor the presence of equally thin A6and A7 mainingFab-7phenotypefromtheprogeny((cid:122)20,000)ofthe
tergites. Out of (cid:122)35,000males, threesuch flies were found. crossbetweenFab-7UC21-10,1-d/TM3SbP(ry(cid:49))(cid:68)2-3malesand
After establishing stocks of these lines, the presence of the Dp(3;1)bxd111/Dp(3;1)bxd111; ry506Abd-BD16/ry506 Abd-BD16 virgins.
Fab-7 deletion was confirmed by Southern blot analysis and Incontrast,allnineputative5(cid:57)deletionsshowedsomedegree
the presence of Abd-BD16 bygenetic complementation test in oftransformationofA6towardA7(Fab-7phenotype)intrans
all three putative recombinants. Having established the use- towild-typechromosomes.Thisphenotypeallowedustosepa-
fulnessofthismethod,weconstructeddoublemutantcombi- rate the chromosomes carrying the new derivatives of the
nations of Fab-7 with Abd-BD14 and UC21-10,1-d in the same insertionalmutationfromthery506Abd-BD16chromosomeand
way. toestablishstocks.Twoofthem,Abd-BPSz1andAbd-BPSz2,showed
R5LR7R:Fortheconstructionofthischromosome,wetook significant complementation over iab-7Sz, i.e., the size of A7
advantageof thefactthat thedesiredproductof recombina- wasreducedcomparedto Abd-BD16/iab-7Sz,.Southernanalysis
tion between the two inversions In(3R)iab-7R7 and In(3R)iab- detectedadeletionof10.8kbbetweenmappositions(cid:49)155.6–
7R5 should contain a deletion in the region 87C1-2;87D1-4, 156.8 and (cid:49)165.6–166.6(3.2 kbdownstream and 7.6 kbup-
includingthekarmoisin(kar)locusat87C8(Gauszetal.1979). streamof the site of the original insertion) in Abd-BPSz1, and
Tp(3;1)bxd111/(cid:49) ; iab-7R5/iab-7R7 females were crossed to cu a 5.5-kb deletion between map positions (cid:49)155.6–156.8 and
kar/cukarmales,andtheprogenywerescoredforkarpheno- (cid:49)160.2–161.3(3.2kbdownstreamand2.3kbupstreamofthe
type.Of 180flies scored,we recoveredsevenkar individuals. siteof theoriginal insertion)in Abd-BPSz2.
Stocks of these putative recombinants were established and
checked cytologically. All of them contained the inversion
In(3R)87C1-2;89E3-4andthe deficiency Df(3R)87C1-2;87D1-4. RESULTS
Isolation of rearrangements that eliminate trans-suppres-
In adult wild-type males the program specifying the
sion:We induced chromosomal rearrangements on the Fab-7
development of the A7 abdominal segment does not
chromosome by irradiating Fab-7/Fab-7 homozygous males
with Xrays(4000rads;1000rads/min,0.5-mmAl filter)and produce a visible tergite or sternite (Figure 1a). This
crossing them to Abd-BD16/TM6B Tb virgins. After 6days the developmental program depends on the normal func-
parents were discarded. Among the male progeny (about tioningoftheiab-7cis-regulatorydomainthatisrespon-
3000) we identified 12 flies in which trans-suppression was
sibleforgeneratingtheappropriatelevelofAbd-BRNA/
weaker or no longer visible. We then tested these new re-
protein expression in PS12/A7. When iab-7 is deleted
arrangements over the Cbx1Ubx1 chromosomes. All but one
showedsignificantreductionoftheweakdominantwingphe- on both homologues, Abd-B expression in PS12/A7
notypegeneratedbytheCbx1mutationthroughthemisexpres- comes under the control of theiab-6 cis-regulatory do-
sion of Ubx(cid:49) on the homologous chromosome. Cytological main, resulting in thetransformation of PS12/A7 into
examinationoftheexceptionalcaserevealedabreakpoint at a copy of PS11/A6 (Galloni et al. 1993). Because of
the BX-C, 89E1,2. By complementation analysis, we showed
thehaplo-insufficiencyoftheAbd-Bhomeoticgene,dis-
thatthebreakpointinactivatedtheiab-4cis-regulatoryregion.
Weisolatedtransvection-disruptingrearrangementsonthe ruptions in the normal developmental program of
Fab-7 Abd-BD16 chromosome ina similar way.IrradiatedFab-7 PS12/A7areevidentinanimalsheterozygousforaniab-
Abd-BD16/TM6C Sb males were crossed to Cbx1Ubx1/TM1 fe- 7mutation,suchasiab-7Sz,whichdeletestheiab-7region
males.Outof(cid:122)1500F1fliesweisolatedfiveindividualsshow- (Gallonietal.1993).Inthiscase, A7assumes aniden-
ing significantly reduced or no Cbx phenotype. Cytological
tityinbetweenthatofthenormalA6andA7.Themixed
examinationofthetwomutationsthatcompletelyeliminated
the Cbx phenotype, TSR-11A Fab-7 Abd-BD16 and TSR-59A identity of A7 is manifested as a rudimentary tergite-
Fab-7 Abd-BD16, showed the presence of the rearrangement likestructureposteriortoA6withacharacteristicshape
T(3;4)89A-B;102A in the first, and Df(3R)88D;89D (cid:49) andsize(Karchetal. 1985). Thisphenotypic transfor-
Tp(3;2)89E;98C;39A inthe secondmutation. mationisshowninFigure1bandsummarizeddiagram-
Isolationofnew deletionsinthe Abd-Bgene by P-element
maticallyinFigure2.AsimilareffectonA7development
remobilization: In order to generate deletions that remove
is also observed in animals heterozygous for an Abd-B
sequencesaroundthepromoteroftheAbd-BclassAtranscrip-
tion unit, theP-elementinsertion UC21-10,1-d,localized 253 mutation;however,inthiscasethedisruptionsindevel-
bpupstreamof theproximalAbd-B promoter(McCall etal. opment are not restricted to A7, and the morphology
1994), was mobilized by introducing the transposase source oftheadjacentsegments A6andA5arealteredaswell.
P(ry(cid:49))(cid:68)2-3.First,theFab-7mutationwasrecombinedontothe
A phenotype exactly the opposite of that produced
chromosome carrying theinsert. [Incontrast to theoriginal
by iab-7 mutations is observed for the gain-of-function
chromosome, these recombinants survive as adult homozy-
gotes, suggesting that the reported lethality (McCall et al. mutationFab-7;PS11/A6is transformedintoPS12/A7.
1994)shouldbeduetosecond-sitemutationslocatedproximal This is due to the ectopic activation of iab-7 in PS11/
1034 L.Siposet al.
A6. Asa consequence,the number of visible segments notype of Fab-7 in A6. This partial suppression is not
in Fab-7 homozygous males is reduced from six to five duetosomeunusualpropertiesoftheAbd-BD16;precisely
(Gyurkovics et al. 1990). Although disruptions in A6 the same A6 phenotype was observed when other Abd-B
development are also found in Fab-7/(cid:49) males, the ex- point mutations (Abd-BS1, Abd-BS4; Tiong et al. 1985)
tentofthetransformationis reducedcomparedtothat were combined with Fab-7 (data not shown).
observed in homozygous mutant animals. Instead of These observations could mean that the activity of
the PS12/A7 identity seen in homozygotes, PS11/A6 Abd-B is “more haplo-insufficient” in A6 than in A7. If
assumesamixedPS11/A6-PS12/A7 identityin animals this were true, we would expect to observe a similar
heterozygousfortheFab-7mutation.Infact,themixed suppressionoftheFab-7phenotypeinA6bythedeletion
identity phenotype of PS11/A6 observed in Fab-7/(cid:49) Df(3R)P9, which removes the entire BX-C, including
males closely resembles the mixed identity phenotype the Abd-B gene.However,as was observedfor theFab-7/
ofPS12/A7observediniab-7(cid:50)/(cid:49)(orAbd-B(cid:50)/(cid:49))male iab-7Sz combination, segments A6 and A7 in Fab-7/
flies(see Figure 1, c and b). Df(3R)P9malesshowthesameintermediateA6-A7phe-
The incomplete transformation of PS11/A6 in notype (see Figure 1f). Similarly, the combination of
Fab-7/(cid:49) males could be attributed to a haplo-insuffi- Fab-7 with a deletion, Df(3R)R59, which removes both
ciencyforiab-7.InFab-7/(cid:49)males,theiab-7cis-regulatory iab-6 and iab-7 but not the Abd-B gene, has the same
domainontheFab-7-containinghomologue isactive in phenotypeas Fab-7/iab-7Sz(datanotshown), indicating
PS11/A6, whereas the iab-7 cis-regulatory domain on thatthefunctioningofiab-6intransisirrelevantforthe
thewild-typehomologueisnot.Ifregulationonlyoccurs phenotype of Fab-7 in A6.
in cis, Abd-B expression in PS11/A6 should be driven Chromosomal rearrangements eliminate the effect
byinteractions betweentheiab-7 cis-regulatorydomain of the Abd-B point mutations: The suppression of the
andtheAbd-BgeneonlyontheFab-7homologue.Such Fab-7 gain-of-function phenotype by Abd-B point muta-
interactions should not occur on the wild-type homo- tions in trans implies that our original assumption,
logue in PS11/A6 because iab-7 is inactive. This inter- namely,thatregulatoryinteractionsonlyoccur incis, is
pretation is supported by the phenotype of Fab-7/iab- incorrect. Instead, it suggests that some type of trans-
7Szmutantanimals.Inthisgenotype,onlyasinglecopy regulatoryinteractionor“transvection”mustalsooccur.
oftheiab-7cis-regulatorydomainshouldbeavailableto Inparticular,atrans-regulatoryinteractioncouldpoten-
drive Abd-B expression, not only in A6 and butalso in tially explainwhy theFab-7 gain-of-function phenotype
(cid:49)
A7. In males carrying this mutant combination, both ofFab-7/Abd-B animals isstronger thanthatof Fab-7/
(cid:50)
PS11/A6 andPS12/A7would be expectedto assume an Abd-B animals; in the former case, the Abd-B genes
identityinbetweenthatofPS11/A6andPS12/A7.This in cis and in trans would produce functional products,
is the case. Moreover, as illustrated in Figure 1d and whereas in the latter case, functional products would
Figure2,segmentsA6andA7intheFab-7/iab-7Szmales onlybe produced by the Abd-B in cis.
closelyresembletheA7segmentfoundiniab-7(cid:50)/(cid:49)(Fig- Transvection is classically tested by generating chro-
ure 1b)males. mosomalrearrangementsthatdisturbhomologouspair-
Taken together,these results would seemto suggest ing.Forthispurpose,wefirstscreenedforX-ray-induced
that,iftherestoftheAbd-Bdomainremainsunchanged, mutationsontheFab-7chromosome,whichreducedor
thephenotype/identityofPS12/A7(orPS11/A6inthe eliminatedthesuppressionoftheFab-7gain-of-function
Fab-7mutant) directlyreflectsthe number ofactiveiab-7 phenotypebyAbd-BD16,thatis,forFab-7*/Abd-BD16males
cis-regulatorydomains. inwhich theA6 tergitemore closely resembledthe A7
Fab-7issuppressedbyAbd-Bpointmutationsintrans: tergite. [We used Abd-BD16 as a representative allele in
We nextexamined theeffects of combining Fab-7 with thisandsubsequentexperimentsbecauseitisamolecu-
anAbd-Bpointmutation,Abd-BD16.Ifourhypothesisthat larly characterized mutation that does not code for a
the identity of PS11/A6 and PS12/A7 depends upon detectableprotein(Bouletetal.1991).]Outof(cid:122)3000
the number of iab-7 domains that are active in these mutagenized chromosomes,we recovered 12 indepen-
twoparasegmentsis correct, then Fab-7/Abd-BD16 males dentFab-7*/Abd-BD16malesin whichtheA6tergite was
wouldbeexpectedto havethesameintermediate phe- as thin or nearly as thin as the A7 tergite. For further
notype in A6 as was observed in Fab-7/iab-7Sz males. analysis, we established stocks of these 12 Fab-7* chro-
However, as illustrated in Figure 1e and Figure 2, this mosomes.
is not the case. Although A7 has the same phenotype Chromosomalrearrangementsthatinterferewiththe
inthetwomutant combinations,thephenotype ofseg- postulatedtransvectionbetweenFab-7 andAbd-B might
ment A6 in Fab-7/Abd-BD16 males differs from that in also beexpected to disrupt pairing-dependent interac-
Fab-7/iab-7Szmales.TheA6tergiteintheFab-7/Abd-BD16 tions elsewhere in BX-C. A well-known example of a
genotypeisenlargedrelativetotheA7tergite,andmore pairing-dependentregulatoryinteractioninBX-Cisthe
closelyresembles the wild-typeA6 segment.This unex- wing transformation induced by the Cbx1Ubx1 chromo-
pectedfindingsuggeststhat,unlike iab-7Sz,theAbd-BD16 some when it is paired with a wild-type copy of BX-C
mutationpartially suppressesthe gain-of-function phe- (Lewis 1955,1985).The12 Fab-7* chromosomes were
Transvection inthe Abd-B Domain 1035
Figure 1.—Abdominal cuticles ofadult males.(a) (cid:49)/(cid:49). In wild type,A7 is represented onlyby a pairof tracheal openings
(arrowhead). (b) iab-7Sz/(cid:49). The haplo-insuffiency of the iab-7 cis-regulatory region results in the addition of a rudimentary,
seventh tergite. (c) Fab-7/(cid:49). Ectopic activationof iab-7 in A6 reduces the sixth tergite to the size of the seventhin iab-7Sz/(cid:49).
(d) Fab-7/iab-7Sz. Because only a single iab-7 is active in A6 and A7 (that of the Fab-7 chromosome), both sixth and seventh
tergites are reduced to the size of the seventhin iab-7Sz/(cid:49). (e) Fab-7/Abd-BD16. A7 is similar to that of the previous genotype,
but the A6tergite isenlarged. (f)Fab-7/Df(3R)P9. If, in addition to iab-7, the transcopy of Abd-B is also deleted,both A6 and
A7 tergites areagain similar to those of the genotype Fab-7/iab-7Sz.(Additionally, due to the partial transformationof A5 into
A4, lack of dark pigmentationin patches of A5is alsoevident.) (g) Fab-7/McpB116iab-7SzAbd-BD16. Thesize of thesixth tergite is
similar to that of thesixthtergite in Fab-7/Abd-BD16 males e,indicating that the presenceor theabsence of an inactivecopy of
iab-7 in trans is indifferent for the suppression of the Fab-7 phenotype. However, A7 is enlarged to the size of A6 in Fab-7/
McpB116iab-7SzAbd-BD16 trans-heterozygotes. As shown in h, if Fab-7 is replaced with a wild-type chromosome in this genotype
(McpB116iab-7SzAbd-BD16/(cid:49)), A6 becomes similar to wild type, but A7 remains about the same asin Fab-7/McpB116iab-7SzAbd-BD16,
indicating that the Fab-7 deletion only ectopically activates the wild-type iab-7 in PS11, but does not alter its ability to trans-
regulateAbd-B.
1036 L.Siposet al.
TABLE1
Listofbreakpointsofrearrangements thatreducetrans-suppression inFab-7/AbdD16males
Cytologyin the Effect on
TSR-XSz, Fab-7 criticalregion trans-suppression Note
TSR-1Sz, Fab-7 T(2;3)2R heterochromatin; 89B (cid:49)(cid:49)(cid:49)
TSR-2Sz, Fab-7 heterochromatin(?);87B (cid:49)(cid:49)
TSR-3Sz, Fab-7 T(2;3)2R heterochromatin, 87F-88A (cid:49)(cid:49)
TSR-4Sz, Fab-7 In(3R)heterochromatin; 90E-F (cid:49)(cid:49)(cid:49)
TSR-5Sz, Fab-7 T(2;3)26A; 3Rheterochromatin (cid:49)(cid:49)
TSR-6Sz, Fab-7 T(2;3)28E;87B8,9 (cid:49)(cid:49)
TSR-7Sz, Fab-7 T(2;3)59B-C;81F (cid:49)(cid:49)
TSR-8Sz, Fab-7 In(2R)81F;88B (cid:49)(cid:49)
TSR-9Sz, Fab-7 T(2;3)58F;3R heterochromatin (cid:49)(cid:49) homozygousviable
TSR-10Sz, Fab-7 In(3LR)62B; 81F (cid:49)(cid:49) homozygousviable
TSR-11Sz, Fab-7 T(Y;3R)87B (cid:49)
TSR-12Sz, Fab-7 In(3LR)79E-80B; 89E (cid:49)(cid:49)(cid:49) iab-4(cid:50)
Additionalrearrangements outsideofthecriticalregionare presentin TSRSz(cid:50)1,3,5,6,8and12;onlythe
relevant breakpointsare listed.(cid:49)(cid:49)(cid:49),strong effect; (cid:49)(cid:49),moderateeffect;(cid:49), weakeffect.
testedfortheirabilitytointeractwiththeCbx1Ubx1chro- maininPS11/A6islimiting;otherwise,thepresenceof
mosome, and we found that all but one significantly a competing Abd-B point mutant in trans to the Fab-7
suppressed the wing phenotype induced by the Cbx1 chromosomewouldhavenophenotypicconsequences.
mutation.Moreover,thestrengthofthephenotypicef- This supposition—that the regulatory capacity of iab-7
fectsofthese11Fab-7*chromosomescloselyparalleled islimiting—seemstobecorrect,becausethephenotype
thatfoundincombinationwithAbd-BD16:mutationsthat of PS11/A6, when the Abd-B gene in trans is either re-
exertedastrongeffectontheFab-7/Abd-BD16phenotype movedentirely(asinFab-7/Df(3R)P9)ornolongeravail-
alsostronglysuppressedthewingphenotype(notshown). able for pairing (as in TSR,Fab-7/Abd-BD16), is indistin-
AsexpectedfromthesuppressionoftheCbx1pheno- guishable from Fab-7/(cid:49).
type,cytologicalexaminationofthese11Fab-7*chromo- Competition between the iab-7 cis-regulatory do-
somesrevealed thattheyallhadrearrangementsaffect- mains: In the experiments described in the previous
ingtherightarmofthethirdchromosome.Theposition section, nonproductive trans-interaction between the
ofthebreakpoints forthe11Fab-7*chromosomes that iab-7 regulatory domain on the Fab-7 homologue and
suppress the Cbx1 phenotype (TSR-1Sz,Fab-7 to TSR- theAbd-Bpointmutationontheotherhomologuesup-
11Sz,Fab-7)arelistedinTable1.All11ofthemhaveone pressedthegain-of-functionphenotypeinA6.Wespecu-
breakpoint between BX-C and the centromere, and a lated that the trans-interactions between iab-7 and the
secondbreakpointoutsideofthisregion.Thisarrange- mutant Abd-B gene were permitted in A6 because the
ment of breakpoints follows the rules established by iab-7 regulatory domain on the homologue containing
Lewis(1955)forchromosomalaberrationsthatdisrupt themutantAbd-Bgeneisnotactiveinthissegment(see
transvection in BX-C.The one exception,TSR-12Sz,Fab-7, Gallonietal.1993).Incontrast,suchtrans-interactions
provedtobean iab-4mutation, andit hasaninversion might not be as strong in A7 because cis-interactions
breakpoint in BX-C (see Table 1). Although this re- betweentheAbd-Bgeneandtheiab-7regulatorydomain
arrangementshoulddisruptpairingintheAbd-Bregion onthesamehomologuewouldreducetheabilityofthe
of BX-C, it would not be expected to affect pairing in Abd-B promoter to interact in trans. This model could
theUbx region of BX-C. potentially explain the phenotypic difference between
TheseresultsarguethatthesuppressionofFab-7gain- A6 andA7 in Fab-7/Abd-BD16 males (see Figure 2).
of-function phenotype by Abd-B point mutations is If this model is correct, cis-competition in segment
likely to be due to a nonproductive trans-regulation. A7shouldbeeliminatedbydeletingtheiab-7regulatory
Whena wild-type geneis present onboth homologues domain on the Abd-BD16-containing chromosome. The
(Fab-7/(cid:49)), both cis- and trans-regulatory interactions lackofcompetitionfromtheiab-7domainincisshould
can contribute to the level of ABD-B protein that is result in an increase in the frequency or strength of
ultimatelyexpressed inPS11/A6.Although thesesame trans-interactions between the iab-7 domain on the Fab-7
cis- and trans-interactions would also occur in Fab-7/ chromosome andthe mutant Abd-B gene onthe other
Abd-BD16 flies, functional Abd-B protein would only be homologue.
producedbytheAbd-BgeneontheFab-7chromosome. Totestthisprediction, werecombinedan iab-7dele-
It should be noted that this model requires that the tionmutant,iab-7Sz,ontotheAbd-BD16chromosome.The
regulatorycapacityoftheectopicallyactivatediab-7 do- phenotypeof(cid:49)/iab-7SzAbd-BD16andFab-7/iab-7SzAbd-BD16
Transvection inthe Abd-B Domain 1037
malesisshowninFigure1handFigure1g,respectively. twoactiveiab-7 domains.Inthesecond,cis-interactions
Two findings are of interest. betweentheiab-7regulatorydomainandtheAbd-Bgene
First, the gain-of-function phenotype of Fab-7 in A6 onthesamehomologuewould,becauseofcompetition,
is stillsuppressedinFab-7/iab-7SzAbd-BD16males(Figure tend to reduce or suppress trans-interactions between
1g),andthissegmentcloselyresemblestheA6segment thisAbd-Bgeneandtheiab-7 regulatorydomainonthe
foundinFab-7/Abd-BD16males(Figure 1e).Thesimilar- other homologue. When there is no competing active
itybetweenthesetwomutantcombinationswouldargue regulatory domain in cis, the Abd-B gene will be more
thattheiab-7cis-regulatorydomainontheAbd-BD16chro- readilyavailabletoengageintrans-interactions.Itisnot
mosome does not contributeto thesuppression of the possible to distinguish between these two models with
Fab-7phenotypeinA6andwouldbeconsistentwiththe thegenetictools presently available; however,we favor
idea that the iab-7 cis-regulatory domain is normally thesecond model, as ouranalysis ofthe effectsof pro-
inactive inthissegment (Gallonietal. 1993).Second, moter deletions (see below) suggests that the balance
segment A7 inboth(cid:49)/iab-7SzAbd-BD16 (Figure 1h)and betweencis-andtrans-interactionscanbeshiftedbycom-
Fab-7/iab-7SzAbd-BD16 (Figure 1g) males is even more petition.
strongly transformedtowardanA6identitythan itisin Mutations that delete sequences near the 5(cid:57) end of
(cid:49)/Abd-BD16 or in Fab-7/Abd-BD16 males. In fact, A6 and the Abd-Bgene enhancetrans-regulation: Theability of
A7 are indistinguishable in Fab-7/iab-7SzAbd-BD16 males the iab-7 regulatory domain to interact with the Abd-B
and closely resemble the appearance of A6 in Fab-7/ genesincisandintransisnotequal.Ifthecis-andtrans-
Abd-BD16 and A7 in (cid:49)/iab-7SzAbd-BD16. It should be em- interactions were symmetrical, the phenotype of Fab-
phasized thatthistransformationin A7identitycannot 7(cid:49)/(cid:49)Abd-B and Fab-7Abd-B/(cid:49)(cid:49) would be identical.
be attributedsolely tothe lack ofiab-7 andAbd-B func- However, thisis clearlynot the case. Incontrast to the
tionontheiab-7SzAbd-BD16chromosome.Thus,thetrans- modestbutdetectabletransformation ofA6toward A7
formationofA7towardA6inFab-7/iab-7SzAbd-BD16males in Fab-7/Abd-BD16 flies, we see no trace of an A6 to A7
ismuchmoreextremethanitiswhenFab-7iscombined transformation in Fab-7Abd-BD16/(cid:49) flies. Similarly,
with deficiencies e.g., Df(3R)P9, Figure 1f, or Df(3R)C4, Abd-BD16 does not complement iab-7Szto any detectable
not shown, which completely remove both iab-7 and degree in A7 (Figure 6a).
Abd-B (compare Figure 1g and f). As expected, if the We reasoned thatit might be possibleto strengthen
Fab-7chromosomeisreplacedbyitsrearrangedversion the interaction of iab-7 with the trans copy of Abd-B by
inFab-7/iab-7SzAbd-BD16,i.e.,inTSR,Fab-7/iab-7SzAbd-BD16 weakening its interactions with the Abd-B gene in cis.
(not shown), the phenotype of A6 and A7 becomes Because the regulatory interactions of iab-7 with the
indistinguishable from that of TSR,Fab-7/Abd-BD16 or Abd-B gene are likely to be mediated, at least in part,
Fab-7/Df(3R)P9(showninFigure2andFigure1f,respec- by sequences in the vicinity ofthe Abd-B promoter,we
tively). thought that it might be possible to weaken the cis-
These findings indicate that trans-regulatory interac- interactionsbysubstitutingAbd-Bpointmutantswithan
tionsin segmentA7 havenophenotypic consequences Abd-B 5(cid:57) deletion mutant, Abd-BD14 (m(cid:50); Karch et al.
if an iab-7 domain is present on the chromosome con- 1985),whichlackssequencesaroundtheproximalpro-
taining the mutant Abd-B gene. To demonstrate that moter (Boulet et al. 1991). As anticipated, we found
this domain must not only be present but also active, thatAbd-BD14weaklycomplementstheiab-7loss-of-func-
we recombined Fab-7 with the Abd-B point mutation tionmutant iab-7SzinA7(Figure 6b).Moreover,unlike
Abd-BD16 and then examined the phenotype of Fab-7/ inFab-7Abd-BD16/(cid:49),wewereabletodetectaweaktrans-
Fab-7Abd-BD16 males. In these animals, both copies of formationofA6intoA7inFab-7Abd-BD14/(cid:49)adults.This
iab-7willbeectopicallyactivatedinA6.Ifwearecorrect phenotype of weak trans-interaction is best seen in the
in assuming that the regulatory domain in cis must be sixth sternites of females (see Figure 3a).
active in order to reduce trans-regulatory interactions Toruleoutthepossibilitythat thisweaktransforma-
with themutant Abd-Bgene,thenthephenotype ofA6 tion is due to a “leakiness” of the Abd-BD14 mutation,
andA7intheseanimalsshouldbeidenticalandresem- we examined thephenotype of Fab-7Abd-BD14/Df(3R)P9
ble theA7segment inFab-7/Abd-BD16 animals. Indeed, hemizygotes and Fab-7Abd-BD14/Fab-7Abd-BD14 homozy-
this is the case (see Figure 2). gotes. In neither case did we detect transformation of
Two different(but not mutually exclusive) hypothe- A6intoA7(notshown).Infact,itappearsthattheweak
ses could explain the phenotypic difference between transformationobservedinFab-7Abd-BD14/(cid:49)animalsre-
the segments in which one vs. two iab-7 regions are quiresthatthewild-typecopyoftheAbd-Bgeneintrans
active, e.g., the A7 of Fab/iab-7Sz Abd-BD16 and Fab-7/ can pair. Thus, transformation is not detectable when
Abd-BD16. The first postulates that the iab-7 regulatory pairingisefficientlydisrupted(Figure 3b).Conversely,
domainsengagenotonlyin cis-butalsointrans-regula- trans-interactioncanbedetectedinbothFab-7Abd-BD14/
tory interactions (indicated by dotted arrows in Figure iab-7Sz and Fab-7Abd-BD14/Df(3R)R59 flies (not shown),
2).Inthiscase,theresultingphenotype dependsupon where the trans copy of the iab-7 regulatory domain is
thesumoftheinteractionscontributedbyeitheroneor deleted but the Abd-B gene is intact.
1038 L.Siposet al.
Figure 2.—Diagram-
matic recapitulation of the
most relevant results (pre-
sented in part in Figure 1)
and their interpretation.
Continuous line represents
theactiveformofiab-6and
iab-7; shadedrectangle,the
inactive (closed) form of
iab-7; small black dots, the
Fab-7 and the putative Fab-8
boundaries; vertical lines,
the endpoints of Fab-7 and
iab-7Sz deletions; filled cir-
cle,theproximalAbd-Bpro-
moter; bold arrow, the
Abd-B class A transcription
unit;andX,thepresenceof
apointmutation.Theregu-
latory interaction between
Abd-B and iab-7 is repre-
sented by curved arrows.
Dottedarrowsandquestion
marks symbolize the possi-
bilityofmutualtrans-regula-
tion when iab-7 cis-regula-
tory regions are intact and
activeonbothhomologues.
Thewidthofblackoblongs
in column 2roughly corre-
spondstothesizeoftheter-
gites of the different geno-
types.
OtherlinesofevidencealsosuggestthattheAbd-BD14 trans.Thesedifferencesarepairingdependent,because
promoter deletion mutant is a weaker competitor for the phenotypes of Abd-BD14/TSR,Fab-7 (Figure 4c) and
interactions with iab-7, either in cis or in trans, than a Abd-BD16/TSR,Fab-7fliesareindistinguishable(notshown).
structurallywild-typeAbd-Bgene.First,thetrans-suppres- IsolationofnewAbd-Bmutationswhichenhancetrans-
sionoftheFab-7gain-of-functionphenotypeinsegment regulation by iab-7: The observation that the small
A6 of Abd-BD14/Fab-7 males is significantly weaker (but Abd-BD14deletionenhancesiab-7 trans-regulationpoints
still detectable) than in the combination of Fab-7 with to sequences in the vicinity of the proximal Abd-B pro-
any of the Abd-B point mutants (compare Figure 4, b moteraspotentialtargetsforcis-(andtrans-)regulatory
and a). Second, the haplo-insufficient phenotype of interactions.However,thefindingthatAbd-BD14stillsup-
Abd-BD14/(cid:49) in A7 is clearly weaker than that of Abd-B presses Fab-7 in trans to some extent (compare Figure
point mutations (compare Figure 4, b and a), which 4,bandc) suggeststhatadditionalsequencesmayalso
suggestsahyperactivationofthewild-typeAbd-Bgenein berelevant for trans-regulation.
Transvection inthe Abd-B Domain 1039
Figure3.—Adultcuticlesof (a)Fab-7Abd-BD14/(cid:49),(b) TSR-10Sz,Fab-7Abd-BD14/In(3LR)TM3,SbSer females.Whenpairingof the
homologues isnormal a,A6is weaklytransformed towardA7asevidencedbythe presence ofcharacteristics thatare normally
foundon thewild-type A7,such ashairs pointing towardthemiddleofthe sixthsterniteandthe absenceof trichomesaround
thesternite(indicatedbyarrowheads).Whenpairingisefficientlydisrupted(bytherearrangementsTSR-10SzandIn(3RLR)TM3),
this mild Fab-7phenotypeisnolongerdetectableb. Inaddition,thehaplo-insufficientphenotypein A7isclearlystronger(the
size oftheseventhsterniteisenlarged)thanina,indicatingthatthehyperactivationof theAbd-B geneisalsoeliminatedinthe
absence of somaticpairing.
To further characterize sequences from the Abd-B greatest in the largest promoter deletion, that is, Fab-
genethatcontributetotheseregulatoryinteractionswe 7Abd-BPSz1/(cid:49) (cid:46) Fab-7Abd-BPSz2/(cid:49) (cid:46) Fab-7Abd-BD14/(cid:49) (cid:46)
took advantage of a hypomorphic Abd-B mutation, Fab-7Abd-BD16/(cid:49). Interestingly, precisely the same rela-
UC21-10,1-d,whichhasaP-transposonatposition(cid:49)159 tionship is observed for the complementation of the
on the molecular map (McCall et al. 1994) just up- iab-7deletion,iab-7Sz,inA7.Thus,thestrongestcomple-
stream of the transcription start site of the Abd-B class mentation is observed in Fab-7Abd-BPSz1/iab-7Sz flies,
Atranscript.Byitself,thisinsertionalmutation,likethe which is followed by Fab-7Abd-BPSz2/iab-7Sz, Fab-7Abd-
promoter deletion in Abd-BD14, weakly enhances iab-7 BD14/iab-7Sz, andfinally, Fab-7Abd-BD16/iab-7Sz. The simi-
trans-regulation. Thus, Fab-7UC21-10,1-d in trans to a laritybetweenthesetwoassays suggestedthat wecould
wild-typeAbd-BgenetransformsA6towardA7.Thatthis measure the ability of any Abd-B mutation to promote
transformation of A6 to A7 is not due simply to the trans-regulation by examining its complementation of
residual Abd-B activity of the UC21-10,1-d allele, but iab-7SzinA7.Usingthisassay,wetestedthetrans-regulat-
rather arises from trans-activation of the wild-type ing ability of three previously isolated deficiencies,
Abd-B gene, is suggested by the finding that the A6- Df(3R)C4(Karchetal.1985),Df(3R)U110andAbd-BRD18
A7transformationisstronglyreducedwhenFab-7UC21- (Hopmann et al. 1995) that remove the distal part of
10,1-d is combined with the point mutant Abd-BD16 (le- BX-C. As expected, Df(3R)C4, which removes iab-7
thality coveredby Dp(3;1)bxd111). and part of iab-6 in addition to Abd-B, does not show
To identify sequences that contribute to cis- (and any complementation. However, both Df(3R)U110 and
trans-) regulatory interactions, we mobilized the UC21- Abd-BRD18, which leave iab-7 intact, complement iab-7Sz
10,1-dtransposonontheFab-7UC21-10,1-dchromosome to a high degree (Figure 6, d and c, respectively). As
and then selected for excision events that eliminated expected, these two deletions do not suppress the
the remaining Abd-B m activity (see materials and Fab-7 phenotype in trans, and the phenotype of A6 in
methods). We then tested these new Abd-B alleles for Fab-7/Df(3R)U110 and Fab-7/Abd-BRD18 (not shown) is
their ability to enhance trans-regulation of a wild-type similar to Fab-7/(cid:49)(see Figure 1c).
Abd-B. Outof ninenew Abd-B alleles, weidentified two Theapproximate limits of the complementing dele-
mutations (Abd-BPSz1 and Abd-BPSz2) that enhanced the tions are shown in Figure 5. These deletions can be
Fab-7 gain-of-function phenotype inA6 when transtoa ordered in increasing strength of iab-7Sz complemen-
wild-type copy of BX-C more strongly than Abd-BD14. tationasfollows:Abd-BD14(cid:44)Abd-BPSz2(cid:44)Abd-BRD18(cid:35)Abd-
Genomic Southern blotting revealed that Abd-BPSz1 has BPSz1. From thisdata it would appear that trans-regula-
a 10.8-kbdeletionbetweenmapposition(cid:49)155.6–156.8 tion is strongest in the case of deletions that remove
and (cid:49)165.6–166.6, andAbd-BPSz2carries a smaller dele- the largest region of Abd-B DNA on the 5(cid:57) side of the
tionofabout5.5-kbfrom(cid:49)155.6–156.8to(cid:49)160.2–161.3 Abd-Bgene(compareFigure6,bandc).Itis alsoclear
(Figure 5). thatdeficienciesthatstrongly enhancetrans-regulation
Other 5(cid:57) deficiencies also enhance trans-regulation: extend well beyond the region thought to correspond
When we compared the transformation of A6 into A7 tothe proximal Abd-B promoter(Busturia andBienz
in Fab-7 Abd-B(cid:50)/(cid:49) flies, it appeared that the strength 1993).
of the trans-activation of the wild-type Abd-B gene is We note that unlike Abd-B “point mutations,” the
1040 L.Siposet al.
Figure 4.—Abdominal cuticlesof adult malesof the genotypes(a) Fab-7/Abd-BD16, (b) Fab-7/Abd-BD14 and (c) TSR-1Sz,Fab-7/
Abd-BD14. Suppressionof the Fab-7 phenotype bythe deletion mutant Abd-BD14 is significantly weaker(but stilldetectable) than
inthecombinationwiththepointmutationAbd-BD16(comparethesizeofthesixthtergitesina,bandc).Duetothehyperactivation
of thewild-type Abd-B gene, the haplo-insufficient phenotype of A7 is suppressed in b (A7tergite is missing and only a single
hair at the tracheal opening, indicated by arrowhead, shows that it is not completely wild type). When pairing is efficiently
disrupted c,both hyperactivation and trans-suppression are eliminated,and A6and A7tergites are equallythin. The thickness
ofarrows in thedrawingsunder eachof theabdomens indicatesthe relativestrength of cis- andtrans-regulation.
Abd-BPSz2, Abd-BRD18 or Abd-BPSz1 deletions do not show sometimesappears to beclonal even withina hemiter-
any sign of an A7 to A6 transformation when they are gite(Figure6d).Df(3R)U110alsodiffersfromtheother
trans to a wild-type BX-C. This difference presumably Abd-Bdeficienciesinthatitfailstocomplementtheloss-
reflectsthe abilityofthese largepromoterdeletions to of-function iab-7 phenotype of Df(3R)R59, a deficiency
enhancetrans-regulatoryinteractionswiththewild-type that extends proximally from the 3(cid:57) end of the Abd-B
Abd-Bgene.Infact,inthecaseoftheselargerdeletions, geneto thebxdregion. Theonlysignof somecomple-
thehyperactivationofthewild-typeAbd-B geneintrans mentation in Df(3R)U110/Df(3R)R59males is the pres-
is even stronger than that observed in the case of the ence of black pigmentation in tergite A6 and A7, a
smaller promoter deletion, Abd-BD14, because a weak phenotype corresponding to the result of the pairing-
A7 to A6 transformation (the presence of at least a insensitive, long-range interaction described by Hop-
single bristle in tergite A7; Figure 4b) is observed in mann et al. (1995) and Hendrickson and Sakonju
Abd-BD14/(cid:49) males. However, these promoter deletions (1995).Allofthe otherAbd-Bdeletionmutations com-
do show the expected haplo-insufficient phenotype plement the R59 deficiency in A7 to a degree roughly
when they are combined with a chromosome like TSR, similar to that observed for iab-7Sz (data not shown).
Fab-7thatdisruptspairing.Inthiscase,theirphenotype ThelackofthiscomplementationbetweenDf(3R)U110
is indistinguishable fromthatof TSR,Fab-7/Abd-BD14 (see and Df(3R)R59 does not seem to be due to the fact
Figure 4c). thatthesetwodeficiencies overlapin theregionjust3(cid:57)
Df(3R)U110 isanunusual case. Itisthelargest ofthe to the Abd-B gene. Like Df(3R)U110, the deletion
Abd-Bdeficiencies thatstill retainsiab-7, andit extends in Abd-BRD18 overlaps Df(3R)R59 in this 3(cid:57) region, yet
from near the3(cid:57) end of Abd-B into the 90A region. As Abd-BRD18 shows the same degree of complementation
expectedfromthelargesizeofthisdeficiency,itexhibits of Df(3R) R59 as that observed for the Abd-BPSz1, which
by far the strongest enhancement of trans-regulation does not overlap.
whenintranstoiab-7Sz.However,unlikeanyoftheother Themostlikelyexplanationfortheunusualbehavior
deficiencies,theextentofcomplementationbythetrans of Df(3R)U110 is illustrated in Figure 7. In the case
copyofiab-7variesfromhemitergitetohemitergiteand of nonoverlapping deficiency combinations such as