Hopmann, Dianne Duncan Ian DuncanMe' Ubx e4/ In(3LR) Cx, Sb eand stocks established using either In(?L)P+ In(3RlP18, MiUbxe40rIn(3LR)Cx,Sb~asbalanc- ers. This method of screening led

Copyright 0 1995 by the Genetics Society of America

Transvection in the iub-5,6,7 Region of the Bithorax Complex of Drosophila: Homology Independent Interactions in tram

Roberta Hopmann, Dianne Duncan and Ian Duncan

Department of Biology, Washington University, St. Louis, Missouri 63130 Manuscript received July 13, 1994

Accepted for publication November 4, 1994

ABSTRACT The Abdominal-B ( Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities

of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14) . Here we focus on iab5, iab6and iab7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cisregulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the ial+5,6,7 region is able to interact with AM-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. LEWIS has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has been detected in a number of Drosophila genes, transvection of the iab5,6,7 region is exceptional in two ways. First, interaction in transwith Abd-Bdoes not require that homologues share homologous sequences within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis. A second unusual feature of iab5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements. The lack of requirement for local synapsis and the tenacity of transinteraction argue that the iab5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of ial+5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Evidence is presented that transaction of ial+5,6,7 requires, and may be mediated by, the region between distal iab7 and Abd- B. Also, we show that ia&5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected.

IT is now fairly well accepted that the bithorax homeotic gene complex (BX-C) (LEWIS 1978) contains just three protein-coding genes, ultrabithmax ( Ubx) , a b dominal-A ( abd-A) and Abdominal-B ( Abd-B) [SANCHEZ- HERRERO et al. 1985a,b; TIONG et al. 1985; for reviews see DUNCAN ( 1987) ; PEIFER et al. ( 1987). These three genes assign unique identities to all eight abdominal segments (Al-AS) , as well as the third thoracic segment (T3) and segments in the genitalia. Thus, each BX-C gene specifies multiple segmental identities. This remarkable parsimony is achieved by differential segment-specific (actually, parasegment-specific ) ( MARTI- NEZ-ARIAS and LAWRENCE 1985) regulation of each BX- C gene. For example, the identities of segments in the posterior abdomen appear to be determined by the level of expression of Abd-B, which is low in A5 [parasegment (PS) 101 , higher in A6 ( PSll ) , still higher in A7 (PS12) and very high in A8 ( PS13) ( KUZIORA and

Corresponding author: Ian Duncan, Department of Biology, Washing- ton University, St. Louis, MO 63130.

MCGINNIS 1988; SANCHEZ-HERRERO and CROSBY 1988; CELNIKER et al. 1989,1990; BOULET et al. 1991; SANCHEZ- HERRERO 1991). That the level of Abd-B is instructive in defining the identities of segments in the posterior abdomen is suggested by the dose sensitivity of Abd-B; animals carrying only one dose of Abd-B show weak transformations of A5, A6 and A7 to the anterior. The different levels of Abd-B expression are controlled by an array of parasegment-specific regulatory regions, termed iab regions, located between abd-A and Abd-B (LEWIS 1978; KARCH et al. 1985; DUNCAN 1987). The iab regions are located in the same order as the order of parasegments they control and each is named for the abdominal tergite that it affects. Thus, the i ab5 region drives low level Abd-B expression in A5, i ab6 intermediate levels in A6 and i a b 7 higher levels in A7 ( CELNIKER et al. 1990; BOULET et al. 1991; SANCHEZ- HERRERO 1991 ) . Although the iab regions controlling Abd-B expression have not been studied in detail, those controlling abd-A ( iab2- iab4) have been shown to act as parasegment-specific enhancers able to drive appro-

Genetics 139 815-833 (February, 1995)

816 R. Hopmann, D. Duncan and I. Duncan

priate expression of a neutral promoter in germ-line transformants (SIMON et aZ. 1990).

In this report we focus on iab5, iab-6 and iab7 and find that these regulatory regions have some quite unexpected properties. We show these regions are able to specify A5 and A6 identities in the absence of function in cis of all three protein coding genes of the complex. Ultimately, we demonstrate that this capacity results from the pairing-dependent interaction of the iab5,6,7 region with Abd-B+ in trans. Such pairing-dependent interactions were discovered at Ubx by E. B. LEWIS (1954), who named the phenomenon transvection. Transvection is presumably facilitated in Drosophila by the intimate synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has now been documented for a number of Drosophila genes [ for reviews see JUDD ( 1988) ; ASHBURNER (1989a) ; TARTOF and HENIKOFF ( 1991 ) ; WU ( 1993) 1 , the case of iab5,6,7 transvection is exceptional in two ways. First, iab5,6,7 transvection can occur between homologues that share no BX-C sequences in common and does not, therefore, require synapsis of homologous sequences within the BX-C. This is the only case of transvection known not to require local synapsis. Second, we find that iab5,6,7 transvection is remarkably difficult to disrupt. Even heterozygosity for certain rearrangements broken within the BX-C itself fail to disrupt the transinteraction. We provide evidence that the ability of iab 5,6,7 to act in trans requires the presence of a relatively small region located between distal iab7 and Abd-B. Unlike several other cases of transvection, action of iab 5,6,7 in trans is independent of the zeste gene. The exceptional properties of iab5,6,7 transvection probably reflect the great distances over which the iab regions must act in cis to influence their regulatory target at the Abd-B promoter.

MATERIALS AND METHODS

General procedures: All flies were raised at 25" and 50-70% humidity in half-pint bottles on the medium of LEWIS (1960). Unless described here, all genetic variants used are described by LINDSLEY and ZIMM (1992). Salivary gland cytology was by standard methods using 45% acetic acid fixation and lacto- aceto-orcein stain (ASHBURNER 1989b). Adult abdominal cuti- cles were mounted for light microscopy as described by DUN- CAN (1982). Mature embryos were dechorionated and removed from vitelline membranes by manipulation on the surface of Time tape and were mounted in a medium containing Shandon Immumount, saturated chloral hydrate in wa- ter and lactic acid syrup (3.6:5.4:1) and cleared on a warming plate overnight. Antibody staining of embryos was as described in KELLERMAN et al. ( 1990).

Isolation of Micro@haZus revertants: To isolate BX-C deficiencies that enter the complex from the right, we screened for X-ray-induced revertants of the dominant mutation Miow cephalus (Me) , which causes a marked reduction of the eye. Mc maps very close to the right end of the BX-C and is associated with a partial duplication of the Abd-B domain of the BX-C: (see below) . Because the effect of Mc on the eye is due

to a gain of function, new deficiencies can be identified as X- ray-induced Me revertants.

Because deficiencies for the right end of the BX-C are dominant sterile in both sexes, we screened for new deficiencies in genotypes that carried a duplication of the complex. Two screening methods were used. In the first, Mc/ TMl, M i sbd' males were packed in gelatin capsules and exposed to 4000 r of X rays in a Torrex 150 X-ray machine. No filtration was used, and X-rays were delivered at 140 kV. Males were then crossed toDp(3;1)68/Dp(3;1)68;1n(3L)P+ In(?R)P18, M4 Ubx e 4 / In (3LR) Cx, Sb e females [ Dp(3; 1) 68 carries the BX-C inserted into the X heterochromatin] for 5 days, after which the male parents were discarded and the female parents were transferred to new food for an additional 5 days. Because all TM1-bearing progeny in this cross die (because M i / M i and Sb/ sbd' are lethal genotypes) , Me revertants are easily identified by screening for individuals with wild-type eyes. Re- vertants were crossed to Dp(3;1)68; In(?L)P + ln(3R)PlR, Me' Ubx e 4 / In(3LR) Cx, Sb eand stocks established using either In(?L)P+ In(3RlP18, MiUbxe40rIn(3LR)Cx,Sb~asbalanc- ers. This method of screening led to the isolation of the R and RD series of revertants. Because of low fertility in this first screen, a second method was adopted. Males of genotype Dp(?;3)P146, Mc/In(?L)P + In(3R)P18, M i Ubx e4 [Dp(?;3)P146carries a duplication for the region from 89D2 to SOD1 inserted at 64C-E] were irradiated as above and crossed to In (3LR) Ubx", sbd' ss bx3"/ In(3LR) 1;211, M i sbd' females. Because all progeny receiving the Zn(3L)P + In(?R)P18, M i Ubx e' chromosome die in this cross, M c revertants were easily identified by screening for wild-type eyes. Revertants were crossed to In(?LR) Ubx", sbd2 ss bx3"/ TM1 flies, and stocks of each revertant were established using TMl as balancer. Mutations in the RS and RK series were isolated by this second method. Once in stock, all revertant chromosomes were examined cytologically and were tested to deter- mine whether BX-C genes were deleted by scoring the haplo- abnormal phenotypes of Ubx and Abd-B. For all partial BX-C deletions recovered in the RS and RK series, Dp(3;3)P146was removed by recombination and the deficiency chromosomes were maintained in stock heterozygous with Dp(?;3)P5, Sb [Dp(3;;3)P5 is a tandem duplication for the material from 89El-2; 90A] or L)p(3;?)P146 + In(?LR)64A; 84D + In(?R) C, Sb Tb. The latter chromosome, abbreviated as R Sh Tb in this report, was constructed and generously provided to us by Dr. E. B. LEWIS.

Isolation of new abd-A alleles: To recover new abd-A alleles on an Abd-B"'" chromosome, Abd-B""/ R Sb Tb males were treated with ethylmethanesulfonate (EMS) as described by LEWIS and BACHER ( 1968) and were crossed to T ( Y;2;3) S 6 " / D f(?R) P9females. T( Y; 2;3) S67' was discovered and very generously provided to us by E. B. LEWIS. It is associated with a strong iab-2 allele and is duplicated for Abd-B+. Thus, new abd-A alleles could be recognized by their i&2 phenotype when heterozygous with T ( Y;2:?) S67', and the sterility of Abd- B"'"was covered in such heterozygotes by the Abd-B+ duplication of T(Y;2;3)Sh".

Generation of double and triple mutants by recombination: The double mutant abd-Ar'24 A6d-B""was recovered from the cross of a t~-A" '~ MC/A~~-B" '~ females to T( Y; 2;3) S"'/ Df(3R)PY males. The desired double mutants were recovered heterozygous with T ( Y;Z;?) Sh7' and were selected by their iab- 2 Me+ phenotype. The triple mutant Ubx' abd-AtJZ4 Abd-B"" was recovered from the cross of FMA3, y2; SMl/ +; ln(3L)P, ss Ubx' Mg Me/ abd-A')24 A6d-BDiX females to Dp(3; 1) 68; ln(3- LR) Ubx''/ In (3LR) Cx, Sb males [ Dp (3; 1) 68 is a duplication of the BX-C carried on the X chromosome]. The desired triple mutants were recovered as males heterozygous with ln(3-

iab5,6,7 Transvection 817

LR) Ubx" and were recognized by their Ubx MCp' Mc' phenotype. The presence of ~ b d - A " ~ ~ and Abd-BDJ8 was confirmed in appropriate progeny tests.

Construction of Op (3;2)D109: Dp(?;Z)D109 was derived by isolating a deletion of most of the non-EX-C material from a much larger duplication, Dp(?;2)ia&5'"' ( JSARCH et al. 1985), which carries the material from 86E through 89E inserted into the heterochromatin of chromosome 2. Because the distal breakpoint of T p ( 2 ; 3 ) i ~ b 5 ~ " is in the iab5 region of the EX-C ( KARCH et al. 1985), Dp(?;Z) Za&5'" carries all BX-C material from Ubx+ through iab4+. Dp (?; 2) iab-5 "" was not used directly in our experiments, however, because its large size causes low viability and because of concern that the results obtained could be due to nonspecific effects of aneuploidy. To remove extraneous material, males of genotype Tp(2;3) iab5'*'/ red sbd2 U b ~ " ~ ~ w e r e irradiated with 4000 r as described above and crossed to red tu-c sbdz 81' females. Deletions of red+ from Dp(3;Z) iab5"' were recovered by se- lecting phenotypically red, Ubx' progeny. The smallest duplication derivative recovered, Dp(?;2)DlO9, carries material from 89A through 89E inserted into chromosome 2 heterochromatin. Dp(3;2)D109 is associated with a translocation [ T (2;3) 35D; 62D] from which the duplication has not been separated. Our failure to recover crossovers between Dp(?;2)D109 and T(2;3)L~w""~' suggests Dp(3;2)DIO9 may also be associated with a T ( 2;3) having heterochromatic breakpoints.

Isolation of transvection-disrupting rearrangements As shown by LEWIS ( 1955), Cbx Ubx/ ++ heterozygotes show a weak Cbx phenotype that apparently results from the pairingdependent tramactivation of Ubx' by Cbx. Rearrangements that disrupt somatic pairing in the vicinity of the EX-C can be detected by their suppression of this transactivation. Ac- cordingly, males carryin Abd-BD", Df(?R) ull0 or the double mutant abd-ADZ4 Abd-BDI5 (see below) were subjected to 4000 r of X rays and crossed to females of genotype Dp (3; I ) 68/ Dp(3; 1) 68; Cbx Ubx gl'/ Cbx Ubx g13 (stock kindly provided by E. B. LEWIS). Flies showing suppression of Cbx (recognized by normal wing position) were selected and retested by cross- ing to Dp (3; 1) 68; Cbx Ubx &/ Cbx Ubx gl'. The large majority of rearrangements detected in this way only partially blocks transvection, as indicated by incomplete development of the alula when heterozygous with Cbx Ubx. Only those rearrangements that show essentially complete alula development in such heterozygotes were chosen for study in this report.

Breakpoints of all transvection disrupting rearrangements were determined by salivary gland chromosome cytology. Breakpoints of R[Df(3R)ul lO] #4 and R[Df(3R)ul lO] #6 were further characterized by in situ hybridization of BX-C A clones to mutant polytene chromosomes essentially as described by CAI et al. ( 1994).

To quantify their ability to disrupt pairing of the BX-C, selected rearrangements were analyzed when heterozygous with wild type in salivary gland nuclei. A single gland was analyzed for each rearrangement. The squash was systemati- cally scanned and an attempt was made to score pairing at 89E in each nucleus encountered. This was continued until 240 nuclei had been successfully scored. I t is unlikely that any great bias was introduced by this method, because we were able to score most nuclei encountered.

Breakpoint mapping: Breakpoints were mapped by South- ern blotting using standard methods. To simplify the analysis, genomic DNA was prepared from homozygotes or hemizygotes for each of the deficiencies. Homo- or hemizygotes for deficiencies that remove Abd-B could easily be identified at the late embryo stage by the presence of sclerotic plates in the

posterior abdomen (LEWIS 1978). The use of homozygous or hemizygous embryos as the source of genomic DNA greatly simplified the mapping of breakpoints as it allowed us to use missing restriction fragments as well as novel restriction fragments as mapping criteria.

Embryos were selected by hand, and batches of 50-200 were homogenized in microtissue grinders in 0.1 ml grinding buffer (0.1 M Tris, pH 9.1, 0.2 M sucrose, 0.05 M Na2EDTA, 0.5% SDS) . The grinders were rinsed with an additional 0.1 ml grinding buffer that was combined with the homogenate. The homogenate was heated at 68" for 30 min and then SDS and protein were precipitated by the addition of 0.03 ml 8M potassium acetate followed by a 30-min incubation on ice. The precipitate was pelleted by a 10-min spin in an Eppendorf 5415 microcentrifuge at maximum speed. The supernatant was transferred to a fresh tube, extracted with pheno1:chloroform and then chloroform and DNA was then precipitated by addition of 0.2 ml absolute ethanol. After 5 min at room temperature, the DNA was pelleted with a 10-min microfuge spin and then rinsed with 70% ethanol and air dried. The pellet was resuspended in 0.05 ml TE pH 8.0.

An amount of DNA corresponding to 100-200 embryos was digested with restriction enzyme and separated on a 0.8% agar- ose gel. The gel was processed and blotted onto nitrocellulose as described by MANIATIS et al. ( 1982). DNA was crosslinked to the filter with a 45sec exposure to W light on a UVP Chromatc-vue Transilluminator (model TM-36). Filters were hybridized essentially as described in MANIATIS et al. ( 1982), using 5X SSC, 5 X Denhardt's reagent, 10% dextran sulfate, 0.1% SDS and 100 pg/ml denatured salmon sperm DNA. BX- C probes were whole A clones from the BX-C walk ( KARCH et al. 1985). Probes were radioactively labeled with "P dATP and dCTP (NEN) using random hexamer labeling ( FEINBERG and VOCELSTEIN 1983, 1984). Unincorporated label was separated from incorporated by passing the labeling reaction through a Sephadex G50 spin column ( MANIATIS et al. 1982). After hybridization, filters were washed to 2X SSC at 65".

Structure of Microcephalus: To understand the structure of the revertants recovered, it was necessary to define the structure of Mc itself. Because Me suppresses the sterility and weak transformations of A7 toward A6 seen in Abd-B/ + adults, Mc has long been suspected to be associated with a duplication for part of Abd-B ( LINDSLEY and GKEIJ. 1968; LEWIS 1978). Evidence that this is the case is provided by analysis of a partial M E revertant, RSI-86, which is associated with an inversion broken at 89E and 98BC. By recovering crossovers between RTl-86 and In(?R) Ubx', an inversion whose distal breakpoint is also at 98BC but whose proximal break is in Ubx, we found that RSl-86 carries Abd-B' functions at both ends of the RS1-86 inversion. That is, we found that the Ubx". RSl- 86' crossover behaves as though it carries the Abd-B region from iab-8' through i ~ b - 9 ~ but no other BX-C material, whereas the reciprocal crossover, RSl -86"UbxX', behaves as though fully duplicated for all Abd-Bfunctions. These observations demonstrate that RSl-86 is duplicated for part of Abd- B and strongly suggest Mc is as well.

The observation that Mc partially covers the haplo-abnor- mality of Abd-B in A7 but not in A5 or A6 suggests that the Mc duplication should carry at least part of iab7' as well as BX-C sequences further to the right. Consistent with this, we find on Southern blots a 9-kb EcoIU fragment unique to the Me chromosome that is detected by a phage clone (A8060) that covers most of the iab7 region ( KARCH et al. 1985; DUN- CAN 1987). This fragment is very likely from the Me duplication breakpoint. It must be derived from the duplicated right end of the complex, because it is absent in all Abd-B- revertants examined. Moreover, this fragment is retained in a


Ubx abd-A iab-5 iab-6 iab-7 Abd-B iab-7 Abd-B MC

iab-7 Abd-B Of RS4-8

Ubx d d - A iab-5 i~b -6 iab-7 )=WVVL DfDl8

FIGURE 1.-Structure of the Microcephalus ( M e ) duplication and selected derivatives. Shown at the top is the inferred structure of Mc. Straight lines indicate BX-C sequences and wavy lines indicate material outside of the complex. The breakpoint of the Me duplication is indicated by a short vertical line. The distance between the duplicated portion of Abd-B and the intact complex is not known. The origins of Df(?R)RS4-8 and Abd-BDI8 are indicated below the Me duplication.

deletion [ Df(?R) UbxRr4-8, Me] induced on Me that appears to delete the normal copy of the complex but leave the right- end duplicated element intact (see Figure 1 ) . Southern blot analysis of DNA from hemizygous embryos shows that Df(?R) Ubxm4-* is broken within a 1.9-kb EcoRI-BamHI fragment extending from about position +131.7 kb to +133.6 kb on the standard map ( KARCH et al. 1985) and is deficient for all BX-C sequences to the left. Df(?R) UbxR.F4-8 does not revert the Mc phenotype and is deficient for all BX-C functions ex- cept those carried by the Me duplication. The orientation of the Me duplication is inferred from the structure of an internal BX-C deletion recovered, RD18. As indicated in Figure 1, RD18 appears to have arisen by deletion of material between one breakpoint in the complete copy of the complex and one breakpoint in the duplicated element. RD18 lacks the 9-kb EcoRI fragment from the duplication breakpoint, indicating that the Me duplication is in direct tandem orientation. This orientation is supported by a second internal deletion, RDl?, that lacks the breakpoint fragment but retains iab-8’ and iab- 9+ function.

RESULTS

From a total of -237,000 flies screened for X-ray- induced reversion of Microcephalus ( M c ) , 154 revertants were recovered. Of these, polytene chromosome cytology was examined for 124, with the result that 86 ( 69% ) proved to be cytologically visible deletions, 27 (22% ) were cytologically normal and only 11 (9% ) were associated with rearrangement breakpoints (5 inversions, 4 translocations and 2 complex rearrangements). Be- cause the deletion revertants may be useful to others for mapping in polytene sections 89 and 90, we have included the cytology of all BX-C- deficiencies recovered in the Appendix. Of importance to this report are revertants that delete Abd-B+ but not U b x f . Such revertants are easily identified because both genes are haplo-abnormal; Ubx- / + flies have enlarged halteres, whereas Abd-B- / + adults show anteriorly directed transformations of the posterior abdominal segments. Accordingly, all revertants were examined when heterozygous with one normal copy of the complex. Nineteen revertants proved to be Ubx+ Abd-B-, and these were further classified as to whether they delete the bxd region o r abd-A+ by examination of the ventral setal belts

TABLE 1

Partial deficiencies of the bithorax complex

Deficiency Cytology BX-C breakpoint

Dl 8

UllO RK8-5 RSl-98 R1 RS2- 78

RD26 RK8- 7 RD31 R6-41

RK7-15

RS3-13 RK8- 1 7

RK8-21 RK1-13 RS4-8 s10”

Wild type

Df( 3R) 89E3-43 9OA2-3 Df(3R)89E;90CD Df(3R)89E;90A Wild type Df(3R)89E;90E Df(3R)89E;90A T(Y;3)Y;89E Df( 3R) 89E; 90DE Df( 3R) 89E; 90D Df( 3R) 89E; 9OA Df(3R)89E;90A In (3LR) 80; 89E+ Df(3R)89E;90C Df(3R)89E;90E Df(3R) 89E; 90D Df(3R)89C3-5;89E34

Right: +163.5-166.5 Left: +148.5-150

+145.5-148.5 +108-112.5 +94.7-99 +82.2-84.5

+60-69 +58.4-63.1 +56.5-58.2 +45.6-49.2 1-45.6-49.2

+26-30 -4.5-10 -7.5-10.5

-16 -16-21

+131.7-133.6 Df( 3R) 89B9-16; 89E34 + 150

‘The Df(3R))slO breakpoint is from KARcH et al. (1990). Our stock of Df(?R))slO is associated with In( 3LR) 75AB; 86AB.

of homozygous or hemizygous mutant first instar larvae (LEWIS 1978; SANCHEZ-HERRERO et al. 198513). Guided by this genetic characterization, the BX-C breakpoints of revertant deletions were mapped by Southern blotting. As described in MATERIALS AND METHODS, this mapping was much facilitated by isolating genomic DNA from embryos homozygous or hemizygous for each revertant chromosome. The BX-C X clones described by U C H et al. ( 1985) (generously provided by SHICE SA- KONJU and DAVID HOGNESS) were used as probes. The salivary chromosome cytology and BX-C breakpoints of the Mc revertant deletions analyzed are summarized in Table 1. In Figure 2 we show the breakpoints of Mc- revertant deficiencies important to this report. Also shown are two deficiencies that enter the complex from the left, Df(3R) UbxR”-’, whose structure is described


~ b . 7 ~ ~ 2 kb-7~10

I

Mcpl

20 40 60 80 100 120 140 160 180 200 I I I I I I I I I I I I I I I I I I I I

H 7sz H 3 " s 3' q p s C l u r A

abd-A RNA iab-2 iab-3 iab-4 iab-5 iabd iab-7 w w.uc W-3 Y CI.0

Abd-B RNAs

Deletions:

Dl8 *-\x .. RS4-8

. SI0 FIGURE 2.-Structure of the abdominal region of the BX-C and of deletions important to this study. The abd-A exons are

redrawn from KARcH et al. (1990) and the structure of Abd-B transcripts is from CELNIKER et al. (1989) and ZAVORTINK and SAKONJU ( 1989) . The iabregions are as mapped by DUNCAN ( 1987) . Solid bars below the abdominal map indicate regions deleted in the indicated deficiencies. Uncertainties in breakpoint position are depicted by dashed lines. The Df(3R)SIO breakpoint is from KARcH et al. ( 1990) and iab7" is from GALLONI et al. ( 1993). All other deficiencies were mapped as described in the text. Also shown are two rearrangement breakpoints important to this report, i ~ b 7 ~ ~ ' ( KARcH et al. 1985) and iab7sfo ( KARCH et al. 1990).

in MATERIALS AND METHODS, and Df(3R) S I 0 ( TIONC et al. 1987), whose BX-C breakpoint was mapped by KARCH et al, ( 1990) . Df(3R) S I 0 is the deficiency segre- gant from Tp(3;Y) i ~ b 7 ~ ' " , a transposition in which the material from 89B through 89E is inserted into the Y chromosome.

One of the first tests carried out with the Mc-revertant deficiencies was an examination of their complementation behavior with a set of deficiencies that enter the complex from the left (see DUNCAN 1987). As described below, the results revealed some very surprising properties of the region including iab5, iab6 and iab 7 (called iab5,6,7 in this report) .

To orient the reader, in Figure 3 we show cuticle from the abdomen of an adult wild-type male and a male heterozygous for wild type and a deficiency for the entire BX-C [ Df(?R)P9], Note that in wild type, tergites (dorsal cuticular plates) of the fifth (A5) and sixth (A6) abdominal segments are darkly pigmented, whereas tergites of more anterior segments are not. A6 differs from A5 in that its tergite has large areas devoid of trichomes and its ventral sternite lacks bristles. Adults carrying only one dose of the BXC show weak anteriorly directed transformations of A5 (recognizable by loss of pigment) and A6 (recognizable by increased trichome density in the tergite and presence of bristles in the sternite). Note also the presence of a small tergite in A7. Development of A7 is normally repressed in the adult male ( SANTAMA-

RIA and GARCIA-BELLIDO 1972) but occurs when A7 is transformed toward the anterior.

The iub-5,6,7 region can act in trans to promote A5 and A6 identities: The conventional view is that the iab 5,6,7 region assigns A5, A6 and A7 identities by ciF regulation of Abd-B ( SANCHEZ-HERRERO et al. 1985b; DUNCAN 1987; CELNIKER et al. 1990; BOULET et al. 1991; SANCHEZ-HERRERO 1991 ) . Results that seemed initially to challenge this view are presented in Figure 4. Shown are abdomens of adult males heterozygous for either Df(3R) UbxRY4-' or Df(3R)SlO and each of our Abd-B- Mc-revertant deficiencies broken in the iab5,6,7region. As anticipated, all heterozygotes shown have seven fully developed abdominal segments rather than the normal six. The total lack of A7 identity in these animals is expected, because Df(3R) UbxRY4-' and Df(3R) S I 0 both remove all BX-C material from the left through and including much or all of iab7. The surprising result, however, is that A5 and A6 identities are present in some of these genotypes: the two most posterior segments (A6 and A7) are pigmented in heterozygotes involving Abd-BD1* or Df(?R)ullO, which retain the iab 5 , iab6 and most or all of the iab7 regions but lack pigment in heterozygotes with larger deficiencies [ Df(?R)RK8-5 or Df(?R)RSl-98] that remove iab6 and iab7. Microscopic examination reveals that both A5 and A6 characters are present in the posterior segments of the Abd-BDZ8 and Df(3R)ullO heterozygotes

820 R. Hopmann, D. Duncan and 1. Duncan

AI , f ~ A2 ' , . , ,/, I . - _ _ ! , ' .,:\, I . . .\ .'

FIGURE 3.-Cuticular structures of the adult male abdomen. All abdomens shown in this report were prepared as described by DUNCAN ( 1982). Briefly, abdomens were pulled from flies, split middorsally and flattened on a microscope slide for examination. In such preparations, ventral structures are centrally located, and dorsal structures are lateral. All abdomens are oriented so that anterior is to the top. ( a ) Abdominal cuticle from a wild-type (Cantons) male. Note that the tergites (located dorsally) of A5 and A6 are darkly pigmented and that the sternite (ventral plate) of A6 is broad and has no bristles (arrowhead). ( b ) Abdomen from a male heterozygous for wild type and a deficiency for the entire BX-C [Df ( j rR)P9] . Note the incomplete pigmentation of the A5 tergite (indicating a transformation toward A4) . the presence of a few bristles in the A6 sternite (indicating a transformation toward A5) (arrowhead) and the development of a small tergite in A7 (indicating a transformation toward A 6 ) .

(see Figure 5 ) . As summarized in Figure 6, these results are remarkable because they demonstrate that the iab 5,6,7 region can function to specify A5 and A6 identities in the absence of Abd-B+ in cis.

In principle, three types of model could explain how the iab5,6,7region is capable of functioning when separated from Ab&. First, iab5,6,7could cis regulate some other gene (presumably abd-A+ or Ubx+) in some way so as to promote A5 and A6 identities; second, iab5,6,7 could regulate Ahd-B' in trans in a pairingdependent fashion ( i.e., via transvection ) (LEWIS 1954) and third, iab5,6,7 could produce transacting products of its own that promote A5 and A6 identities. The first model predicts that the inb-5,6,7 region should lose its ability to promote A5 and A6 identities when flanked by null alleles of abd-A and Abd-B. Accordingly, an nbd-A- allele, abd-ADZ4, was placed in cis to the Abd-B- deficiency Abd- B"" by recombination. The ~bd-A"'~ allele (KARCH et nl. 1985) is a functional null ( BUSTURIA et al. 1989; unpublished observation) but is weakly protein positive by antibody staining ( URCH et al. 1990). Because abd- A - alleles are lethal when hemizygous, the abd-A')24 Abd- B"" double mutant could not be tested directly in heterozygotes with D f ( 3 R ) U ~ X " ~ ~ ~ - ' or Df(3R)SIO. To allow survival of such heterozygotes to adulthood, we constructed a duplication, Dp(3;2 )0109 , that carries all BX-C functions to the left of iab5 (see MATERIALS AND METHODS). As shown in Figure 7a, Dp(3;2)D109; abd-

Ahd-B'"'/ D f ( 3 R ) U ~ X " ~ ' ~ ~ ' adults are phenotypically indistinguishable from A~-B"'X/T)f(3R)UhXNS4-R

A 1)24

adults in abdominal segmentation and show A5 and A6 identities. Similar results were obtained with abd-A"'f'.", a hypomorphic EMS-induced allele recovered in cis to Abd-B"". Thus, the iab5,6,7 region does not promote A5 and A6 identities by regulation of abd-A in cis. To test for possible involvement of Ubx, the null protein negative allele Ubx' (WEINZIERL et al. 1987) was used to construct the triple mutant Ubx' ~ b d - A " ~ ~ Abd-I?')''. Dp(3; 2) D109; Ubx' aM-A')24 Ab"'"/ Df(3R) U~X"~'~-' males also show A5 and A6 identities, demonstrating that these identities do not result from Uhx expression in cis. The triple mutant of CASANOVA et al. (1987),

abd-A"" Abd-B"', was also tested and behaved identically to our triple. Figure 7b presents an important control here: Dp(3;2)0109; Df(3R)RSI-98/ Df(3R) U~X".'~-' males lack A5 and A6 identities, demonstrating that these identities are not promoted by Dp(3;2)D109in the above genotypes but rather depend upon the iab5,6,7 region. The abd-A"24 Ahd-B"'" and Ubx' abd-A')" Ahd-B"" recombinant chromosomes show the expected phenotypes (LEWIS 1978; MORATA d nl. 1983; CASANOVA et nl. 1987) in homozygous embryos (Figure 8), confirming their constitution.

We note that Df(3R) SI0 was not used in the experiments described above or in those that follow because it has low viability when heterozygous with several of our deletions. Although the reason for this is not known, we suspect this is because Df(3R)SlO [unlike Df(3R)- RS4-81 extends into 89B. In our experience, such deletions have reduced viability in general.

,lJbX?bfx/2

in&5,6,7 Transvection 82 1

Df RS4-8 Df SI0 a*

Of RSI-98

Df RK8-5

Df ull0

Df D l 8

FI(;URE 4.-Abdomens from males heterozygous for Mc- revertant deficiencies broken in the in&5,6,7 region and either Ilf(3R)RY4-8or Df(?R)SlO. All animals show full tergite development in A7, indicating transformation of A7 to the anterior. Note that heterozygotes carrying A / X " * or Df(3R)ullO show almost full pigmentation of the A6 and A7 tergites, whereas heterozygotes carrying Df(3R)RK8-5 and Df(?R)RFl-98 do not. In addition, heterozygotes carrying Abd-B-n"'s show a broadening and reduction in bristle number of sternites in posterior segments, indicating partial A6 character. These results are remarkable because they demonstrate that the ia&6, 7region is able to promote A5 and A6 identities in the absence of an Abd-B gene in cis. Note that Df(3R) RY4- 8 and IIf(3R) SI0 have essentially the same phenotypes in the heterozygotes shown. IIf(3R) RY4-8 is used in almost all other experiments in this report because i t has much higher viability than Df(?R)SIO, and because it is inseparably linked to a convenient dominant marker, Mc.

At the oubet, our second type of model, pairingdependent activation of Ahd# in trans by the ia&5,6,7 region ( i.e., transvection) , seemed very unlikely. It was difficult to see how pairingdependent interactions could occur in ~f(3R).S10/ l ) f3R)u110 heterozygotes, because these deficiencies overlap and have no BX-C sequences in common with which to pair. Moreover, although the CASANOVA et al. (1987) triple mutant behaved identically to our triple mutant, it includes an inversion [In (3R) 89E;99F] ( I . DUNCAN, unpublished data), associated with nhd-A"", that would be expected to cause some pairing disruption within the BX-C. As an initial test of transvection, we determined whether

FI(;L'KE: 5.-High magnification lateral view o f ' the A5, A6 and A7 tergites of a male A/~d-~"'"/l)S(?R)SIO heterozygote. Note that the A5 tergite is only partially pigmented and intermediate in character between A4 and A.5. The A6 tergite is largely A6 in character, but regions devoid of trichomes (small hairs) are not as extensive as in the wild-type A6, indicating partial A5 identity. A7 is strongly A6 in character. Df(3R)u110/Df(3R)SlO heterozygotes are similar but show weaker A6 character in A6 and A7.

iab-5 iab-6 iab-7 Abd-B

DfullO .-( A5 and A6 identities

DfSIO - DfRSI-98 4 A5 and A6 identities DfSlO - absent

Flc;me 6.-Diagrammatic recapitulation of the results presented in Figures 4 and 5. As indicated, comparison of the Df(3R)~r110/I)f(?R)SIO and l~(?R)KS1-98/I)J(?R).Sl0 genotypes indicates that the in1+5,6,7 region is responsible Ibr promoting A5 and A6 identities. However, counter to the conventional view, these genotypes demonstrate that id-5,6,7 can promote these identities in the absence of an AM-Ijgene in cis.

822 R. Hopmann, D. Duncan and I . Duncan

alukAM4 Abd-6 aWA+ A M 6 lab4 lab4 lab7 lab4 iab4 lab7

1 ( )“-DfDi8 + Df RSi-98 )-DfRS4-8 )-DtRS4-8

____I Dp Dl09 - Dp Dl09 FIGURE 7.-Test of whether the A5 and A6 identities seen in Figure 4 result from cis regulation of abd-A by ia&5,6,7. ( a )

Abdomen from a Dp(3;2)D109; a6d-Auz4 AM-B”’”/Df(3R)R‘i4-8 male. Note that A6 and A7 are pigmented. Examination of trichome patterns reveals that both A5 and A6 identities are present. As shown diagrammatically below, the phenotype of this animal indicates that ia&5,6,7 can promote A5 and A6 identities in the absence of both abd-A+ and A6d-B’ in cis. ( b ) Abdomen from a Dp(3;2)D109; Df(3R)RSl-98/Df(3R)RS4-8male. This important control genotype demonstrates that Dp(3;2)0109does not by itself promote A5 and A6 identities and indicates that the iab5,6,7 region is responsible for promoting the A5 and A6 identities in a. Substitution of deletions that extend further into the complex and remove a6d-A [e.g., Df(3R)R’i6-41] for Df(3R)RS1-98does not alter the phenotype shown in b.

FIGURE 8.-Posterior segments of double and triple mutant first instar larvae. ( a ) An ~6d-A‘)~‘ AM-B”” homozygote. All posterior setal belts are A1 in character. The henotype is identical to a6d-A- Abd-B- genotypes described previously (LEWIS 1978; MORATA et al. 1983). ( b ) A Ubx’ a6d-ARZf AM-B‘”’ homozygote. The phenotype is essentially identical to that seen in larvae homozygous for a deficiency that includes the entire BX-C [ Df(3R)P9] ( LEMW 1978), with the exception that cuticular structures are slightly better developed. As described previously for BX-C- genotypes, setal belt3 in T3A7 are T2 in character, whereas the A8 setal belt is T1-like. Also consistent with previous descriptions, sclerotized plates develop in the posterior portion of A8 in both a and b (arrowheads).

iab-5,6,7 Transvection

TABLE 2

Rearrangements of Abd-Pl8 and abd-ADZ4 Abd-BD" chromosomes selected by disruption of a x Ubx tramvection

823

Rearrangement Breakpoints

R(Abd-p'8)#l R(Abd-P")#Z R(Abd-P")#3 R(Abd-pI8)#4 R(Abd-B"18)#5 R(Abd-B"")#6 R(abd-A1'24 Abd-P'bj#l R(abd-AUZ4 Abd-B"")#Z R(abd-AUZ4 Abd-P")#3 R(abd-iirjZ4 Abd-PI8)#4 R(abd-Auz4 Abd-B"")#5 R(abd-ADZ4 Abd-PI8)#6 R(abd-A"z4 Abd-P")#7 R(abd-ADZ4 Abd-P")#8 R(abd-A"24 Abd-B"I8)#9 R(abd-ADZ4 Abd-B"'bj#lO R(abd-AUz4 Abd-&"8)#l 1

T( 2; 3) 60D; 88F T( 2; 3) 60E; 83E T(2;3)58AB;81 T(2;3)21A81 T (3; 4) 89B; 102F In (3LR) 79B; 89B T(2;3)41;89B Complex. New order: 61A-63EF/87F-92D/81-87F/63EF-80/92D-100 In (3LR) 63EF 81 In(3R)81;91F-92A T(2;3)40;86D Complex. New order: 100-81/62F-61A/unknown short segment/62F-80 T(2;3)41;86F 86F/undetermined heterochromatin In (3LR) 80; 89B Tp(89A 89E) into undetermined heterochromatin T(2;3)40 or 41;86E + In(2LR)23DEF;53EF

heterozygosity for T(2;?) bwVDP3 [T (2;3) 59D;81F], a translocation shown by LEWIS to cause a strong disruption of transvection in the Ubx domain of the BX-C (LEWIS 1954), influences A5 and A6 identities in T(2;3) b w M k 3 + Df(?R) &Rs4-'/A6d-B"'' or T(2;?) b w V D p 3 + Df(3R) UbxRY4-'/ Df(?R) d l 0 heterozygotes. No effect was seen in either genotype. To quantify the ability of T(2;?)bwVDP3 to disrupt pairing at the BX-C, salivary gland chromosomes of T(2;?) b w V D e 3 / + heterozygotes were examined. Although the BX-C genes are not ex- pressed in salivary glands, this measure of pairing is rea- sonable as synapsis of homologues in salivary gland nuclei is likely similar to that in nonpolytene cells ( SMOLIK- UTLAUT and GELBART 198'7). Because T(2;?)bwVDe3 causes strong disruption of transvection at Ubx (LEWIS 1954), we were surprised to find pairing at 89E (the locus of the BX-C) in 44 of 80 nuclei examined. To test the effects of more severe pairing disruption, additional rearrangements induced on Abd-B"" were selected by their ability to suppress transvection in Cbx Ubx heterozygotes (see MATERIALS AND METHODS). The rearrangements recovered are listed in Table 2 and are referred to below by the designation R () . These rearrangements cause only moderate pairing disruption of the BX-C in salivary gland nuclei. When heterozygous with wild type, the strongest disruption was caused by R ( # 5 ) , which showed pairing at 89E in 16 of 41 salivary gland nuclei examined. However, none of the six rearrangements analyzed had any effect on A5 or A6 identities, even when pairing was further disrupted by heterozygosity for T(2;?) bwVUP3 [ i.e., in R(Abd-B"") / T(2;?) bwVU" + Df(3R) UbxRs4-' heterozygotes].

The tests described above argue against both the first ( cis-regulation of abd-A+ or Ubx+) and second (activa-

tion of Abd-B+ in transvia transvection ) models proposed to explain how the iab5,6,7 region could function in the absence of an Abd-B+ gene in cis. However, these tests leave open the possibility that both mechanisms operate and that either alone is sufficient for the assignment of A5 and A6 identities. To test this, we derived genotypes in which pairing is disrupted by chromosome rearrangements and in which the iab5,6,7 region is flanked by null alleles of abd-A and Abd-B. Eleven chromosome rearrangements induced on the double mutant ~bd-A*'*~ Abd-B"'*were recovered by suppression of transvection of Cbx Ubx. The breakpoints of these are listed in Table 2. Several, including #1, #2, #9and #20, cause very strong disruptions of pairing at 89E in salivary gland chromosomes. R( ~ b d - A " ~ ~ Abd-B"") #1 , for example, showed pairing at 89E when heterozygous with wild type in only 3 of 40 salivary gland nuclei examined. Heterozy- gotes carrylng these rearrangements and the Op(?;2)- D l 09 + Df(3R) UbxRs4-' combination were then derived. All show A5 and A6 identities indistinguishable from those seen in unrearranged ~ b d - A " ~ ~ Abd-B""/ Dp(?;2)D109+ Df(3R) Ub~~';~-'adults. A5 and A6 identities also remain unaffected in T ( 2 ; ? ) b ~ ~ " ~ , Ubx' ~bd-A"'~ Abd-B""/ Dp(3;2)D109 + Df(3R) UbxRs4-' and T(2;?)bwW'"'', UbxMX'2 abd-A" Abd-BM'/Dp(?;2)D109 + Df(3R) UbxKs4-' heterozygotes. Thus, strong reduction of pairing and elimination of the possibility of cis- interaction with Ubx, abd-A and Abd-B does not block the ability of the iab5,6,7 region to promote A5 and A6 identities.

iab-5,6,7 promotes A5 and A6 identities in trans via transvection: At this point in the analysis, we began seri- ously to entertain the third model proposed above, that the iab5,6,7 region encodes trans-acting products of its


own that promote A5 and A6 identities. However, there was reason for hesitation. Although our experiments rule out transvection similar to that characterized in the Ubx region, they leave open the possibility that transvection occurs at iab5,6,7 but is different from other cases described and involves remarkably avid tran5interactions. As shown below, this turns out to be the case.

To test for transvection more rigorously, we induced rearrangements on Df(3R)ullO [ =Df( 3R) 89E3-4;90A2- 31 . The rationale here was that by eliminating a distal region of homology, Df(3R)ulIO should enhance the pairing disruption caused by heterozygosity for rearrangement breaks proximal to the BX-C. Rearrange- ments were recovered by screening for suppression of transvection when heterozygous with Cbx Ubx. Most rearrangements recovered in this way do not completely block transactivation of Ubxf by Cbx and show some reduction of the alula when heterozygous with Cbx Ubx. By selection of the minority of rearrangements that allow full alula development, we were able to obtain 20 rearrangements that cause strong disruptions of pairing at 89E. The breakpoints of these 20 rearrangements are shown in Table 3. All rearrangements were then tested for their ability to promote A5 and A6 identities when heterozygous with Df(3R) U ~ X ' ~ ' ~ - ' . To intensify pairing disruption at the BX-C, all were tested in animals also heterozygous for T(2;3)b7u""" [ i . e . , in RDf(3R)- u11U] / 7'(2;3) b ~ ~ " " ' ~ + Df(3R) U~X""~-' heterozygotes.

Two rearrangements, R[ Df(3R)u110] #4 and R#6, cause a strong reduction of pigmentation in the posterior segments when heterozygous with Df(3R)-

animal to animal. Both rearrangements cause very strong disruption of pairing of the BX-C when heterozygous with wild type in salivary gland chromosomes. R#4 [T (2;3) 40;89E] is broken within the bxd region of the BX-C [as determined by in situ hybridization of the bxd region clone A2206 (BENDER et al. 1983, 1985) to both sides of the 89E break in salivary gland chromosomes] and shows a weak bxd phenotype when hemizygous. When heterozygous with wild type, R#4 showed pairing immediately distal to the translocation breakpoint in only 1 of 51 salivary gland nuclei examined. Even this one paired nucleus may not have been synapsed at the BX-C (at 89E), because visible pairing started at the distal breakpoint of Df(3R)ulIO, in 9OA. R#6 is a very complex rearrangement in which the BX-C doublets (89E1-4) , and little else, are transposed to a location between heterochromatin and 61F. The position of the BX-C in K#6 was not obvious from direct observation and was determined by in situ hybridization of A clones 8004 and 8060 ( ~ C H et al. 1985) to mutant polytene chromosomes. Because the BX-C region of R#6 is difficult to locate by direct observation, we have not tried to quantitate pairing disruption at 89E. However, the nature of the R#6 rearrangement suggests it probably

ubxRs4-x (Figure 9 ) . Pigment is patchy and varies from

causes near complete pairing disruption. Presumably because of enhanced pairing disruption, R#4/ T(2;3)- bwm'" + Df(3R) U ~ X ' ~ ~ ~ ~ ' animals show significantly less pigment than R#4/ Df(3R) U~X'~ '~- ' heterozygotes (see Figure 10d). For unknown reasons, R#6 is lethal when heterozygous with T(2;?) b ~ " " ' ~ + Df(3R) U ~ X ' " ~ - " and so has been examined with Df(3R) U~X".'~-' only. The effects of R#4 and R#6 and other rearrangements described below argue strongly that in the absence of Abd- B+ in cis, the iab5,6,7region promotes A5 and A6 identities by regulating Abd-B+ in trans via transvection.

Three rearrangements (R#I, R#l3 and R#14) cause moderate variegation of pigment in posterior segments when heterozygous with T(2;3) bw1'"3 + Df(3R) Ubx'""' (Figure 10c). All are broken close to the BX-C and cause strong disruption of pairing in salivary gland chromosomes when heterozygous with wild type. R#I, for example, showed pairing at 89E in only 14 of 65 nuclei examined. Nine rearrangements show weak variegation (Figure lob ) . Rearrangements in this class also cause strong pairing disruptions at the BX-C; when heterozygous with wild type (R#/O, for example, showed pairing at 89E in 8 of 40 nuclei examined) but for the most part are broken at greater distance from the BX-C than are rearrangements of the moderate class. One rearrangement, R#l7, seems anomalous in this group. R#17 is a paracentric inversion with breakpoints in 81 and 89E. The latter breakpoint is likely within the bxd region, as R#I 7 is associated with a strong bxd allele. One might have expected R#/7 to behave much like R#4, which also has one breakpoint in the bxd region and one breakpoint in heterochromatin. However, as found by LEWIS (1954), paracentric inversions in 3R have little effect on transvection in the Ubx domain, whereas translocations such as R#4 have a very strong effect. Consistent with this, we find that R#l7 causes only relatively mild pairing disruption of the BX-C when heterozygous with wild type (29 of 43 salivary gland nuclei examined showed some association of homologues immediately distal to the 89E breakpoint). Six rearrangements have no or very little effect on iab5,6,7 transvection. These rearrangements cause relatively mild pairing disruption when heterozygous with wild type in salivary gland nuclei. R#2, for example showed pairing at 89E in 22 of 54 nuclei examined.

A region between distal iab-7 and Abd-B is required for iab-5,6,7 transvection: As shown in Figures 2 and 4, the i d regions in Df(3R)ulIO retain the ability to act in trans, whereas those carried by the deletion with the next most proximal breakpoint, Dj(3R)RK8-5, do not. This indicates the region between these breakpoints is required for trans-interactions with Abd-B. This region is large, however, encompassing most of iab6 and iab7. Fortunately, iab6 and iab7 alleles associated with rearrangement breakpoints (KARCH et al. 1985, 1990;

iab5,6,7 Transvection

TABLE 3

Effect of rearrangements on tramactivity of the iab-5,6,7 region in Df3R)ul10/T(2;3)bwm3 + Df3R)RS48 heterozygotes

825

~~

Rearrangement Cytology

Strong suppression R[Df(3R)ull0]#4" R[Df(3R)ullO] #@

Moderate suppression R[Df(3R)ullO] #I

R[Df(3R)ullO] #13 R[Df(3R)ullO]#14

R[Df(3R)ullO] #8 R[Df(3R)ullO] #9 R[Df(3R)ullO]#lO R[Df(3R)ullO] #12 R[Df(3R)ullO] #15 R[Df(3R)ullO] #16 R[Df(3R)ullO] #I 7 R[Df(3R)ullO]#l8 R[Df(3R)ullO] #19

R[Df(3R)ullO] #2

R[Df(3R)ullO] #3

R[Df(3R)ullO] #5 R[Df(3R)ullO] #7 R[Df(3R)ullO]#ll R[Df(3R)ullO] #20

Weak suppression

No suppression

T( 2; 3) 40; 89E Complex T(Y3). New order: 61A-61F/S9D-63CD/36CD-21 + 100-90A/63 CD-61F/

89E1-4/heterochromatin + 60-36CD/heterochromatin Complex. New order: 61-80/89D-81 + 100-89D/22A40 + 21-22A/undetermined

heterochromatin Tp(3;2)41;88BC;94D T(3;4)88F;101

T( 2; 3) 40; 87C T(3;4)86D/101 + In(3L)67&76AB T(2;3)40;88B + In(3R)SlBC;SGAB T (2; 3) 40; 88C T( 2; 3) 40; 88A T ( 2; 3) 40; 88A In(3R)81;89E T(2;3)41;87B T(2;3)23AB;85C

Complex. New order: 21-43DE/62AB-67DE/78D-67DE/88CD-100 + 60-43DE/ Complex. New order: 60-57F/82E-93F/62B-82E/57F-41/98BCD-100 + 61-62B/93F- In (3LR) 63EF; 8 1 T(Y;3)88CD

T(Y;3)88D

88CD-78D/62AB-61

98BCD/41-21

T(2;3)24F-25&81

Breakpoint shown to be within the bxd region by in. situ hybridization of phage clone A2206 (BENDER et

'Location of the 89E bands was determined by in situ hybridization of the BX-C phage clones A8004 and al. 1983) t o mutant polytene chromosomes.

A8060 (KAKCH et nl. 1985) to mutant polytene chromosomes.

DUNCAN 1987; CELNIKER et al. 1990; GWRKOVICS et al. 1990; CROSBY et aZ. 1993; GAI.LONI et al. 1993) can be used to further refine the mapping. The rightmost of these breakpoints, Tp(3;Y) iab7"'" ( TIONC et al. 1987; GYUR- KOVICS et al. 1990) (also called Abd-B"'") , is broken near +150 kb on the standard map ( KARCH et al. 1990) and has properties entirely consistent with our deletion studies. Because the BX-C breakpoint of iab7s'" is to the right of the Df(3R)ullO breakpoint, one would expect the portion of the complex containing iab5,6,7 in iab 7"'" to be capable of interacting in trans with Abd-B. Figure 11 shows the abdomen from a Tp (3;Y) iab7s'"/ Df(3R)Rs4-8 heterozygote. A6 and A7 show mottled pigmentation similar to that seen in heterozygotes of the strongly mottled class of R[ Df(3R)u110] rearrangements. This is exactly the phenotype expected, because

is a transposition of the material from 89B through 89E into the Ychromosome and would be expected to cause very strong pairing failure when heterozygous with Df(3R)RS4-8. It is worth noting that Tp(3;Y) iab7""/ D f(3R) P9 males also show mottled pig-

i'b 7 5 1 / I

mentation of the posterior segments. In this genotype, as in Df(3R) ullO/Df(3R) SlO heterozygotes, iab5,6,7 a p pears able to interact in trans with Abd-B in the absence of homologous BXC sequences.

A very different result is obtained with an iab7 rearrangement [ In (3LR) iab7MX2] broken - 10 kb to the left of Tp(?;Y)iab7"'", in the interval +139.5-142 kb. In (3LR) iab7MX2 shows essentially no A5 or A6 identities when heterozygous with Df(3R) RS4-8 (see Figure 11 ) or when hemizygous ( SANCHEZ-HERRERO et nl. 1985a; DUNCAN 1987). Although iab7MX2 is a complex rearrangement (with the new order 61-64A/89A-89E/ 89A-64A/ 89E-100) , the tenacity of transinteractions demonstrated above renders it very unlikely that the inability of iab7Mx2 to interact in trans is due solely to pairing disruption. A likelier explanation is that the iab7Ms2 breakpoint separates iab5,6,7 from a specific region required for action in trans. This required region must be located between the iab7Ms2 breakpoint [localized to the interval + 139.5- 142 kb by KARCH et al. ( 1985) ] and the Df(3R) uZl0 breakpoint (in the in-

826 R. Hopmann, D. Duncan and I . Duncan

FIGURE 9.-Heterozygotes of R(ullO)#4 [ =T(2;5)40;89E] and R(u110)#6 [a complex T(Y;J) that includes a transposition of the 89E doublets to a location between 61F and heterochromatin] with DJ(3R) RW-8. Both R#4 and R#hcause near complete disruption of pairing at 89E in salivaly chromosomes when heterozygous with wild type. ( a ) An R(ullO)#4/I)f(3R)R.S4-8 heterozygote. Note that pigment is mottled in A6 and A7. The AI tergite is reduced because R#4 is broken within the bxd region of the BX-C. (b) An R(ullO)#6/ Df(3R) RW-8 heterozygote. This genotype shows the strongest suppression of pigmentation in A6 and A7 we have been able to produce. The suppression of pigmentation in these genotypes and those in Figure 10 indicates that the inb-5,6,7 region can promote A5 and A6 identities by interacting with AM-13 in f m n s .

terval +145.5-148.5 kb) , an area of some 9 kb or less that includes distal inb7 and probably also some material between inb7 and Abd-R.

Transvection of iab-5,6,7 is zest% independent: Be- cause many cases of transvection described require the product of the Zpstegene (Wu 1993; but see PATTATUCCI and KAUFMAN 1991; MARTINEZ-LABORDA et al. 1992), we tested a panel of z s t e alleles for effect on the trans interaction between inb5,6,7 and AM-R. The alleles

These include alleles that suppress or otherwise influence transvection at zu (JACK and JUDD 1979), Ubx ( KAUFMAN et nl. 1973; BARU and BHAT 1981 ; GELRART and Wu 1982), dpp ( GELBART 1982; GELRART and Wu 1982) and y (GEYER et nl. 1990). In our hands, z"""'.~',

completely block transvection in Chx ubx/ ++ heterozygotes, whereas zn and and z06Y-3 cause strong, but incomplete, suppression. None of the above alleles has any effect on A5 or A6 identities in Abd-B"IX/Df(3R) UI)X".'~-" heterozygotes. To test whether rearranged genotypes might provide a more sensitive background, the effects of several z s t e alleles were tested in Df(3R)ulIO/Df(3R) U~X"~'~-~, R(Abd- B"") / Df(3R) U ~ X I ' . ' ~ - ~ and R(nl~i-A"~' Abd-B"") / Dp(3;22)0109 + Df(3R) U~X".'~-* heterozygotes, but again, all results were negative.

tested were Z n , zfldIJXJ z 0 d / J X ) 2 Z n f i Y - 2 p 9 - 3 and z1177/t. , , ,

zn69-2 and zlli7/t

DISCUSSION

In this report, we describe a most unusual case of transvection in the bithorax complex (BX-C) . We find that the iab5,6,7 region, which normally regulates Abd-

R expression in A5, A6 and A7 ( more precisely, parasegments 10-12), is able to interact with Abd-H in /ram so as to specify A5 and A6 identities. This transinteraction is remarkable in at least three ways: the interaction is independent of synapsis within the BX-C, it is extraordinarily difficult to disrupt by chromosome rearrangement and it appears to be independent of the z s t e gene. We present evidence that transaction of iab 5,6,7 requires the presence of the region between distal inb7 and Abd-B.

Our discovery of transaction of inb5,6,7 came about in studies of partial BX-C deficiencies isolated as revertants of the dominant mutation Microccghalus. These deficiencies all remove Ahd-R but extend leftward into the BX-C to differing extents. A surprising observation was made when these deficiencies were examined in heterozygotes with deficiencies that remove Uhx and extend rightward into the complex. Of particular inter- est in this regard are two deficiencies [ Df(3R) U ~ X " . ' ~ - ~ and Df(3R)SIO] that remove Ubx, nbd-A and all in6 regions through inb7 but leave the Abd-B transcribed region intact. In heterozygotes with D ~ ( ~ R ) U ~ X ' ' ' ~ - ~ or I)s(3R)S10, we find that Abd-B- deficiencies that retain the inb5,6,7 region [ Df ( 3 R ) u l l0 or Abd-B"'x] show A5 and A6 identities in their posterior segments (see Fig- ure 4 ) , whereas AM-B- deficiencies that remove most or all of this region [ Df ( 3 R ) RK8-5 or Df(3R) RTI - 981 do not. That is, we find that the inb5,6,7 region of the BX-C is able to promote A5 and A6 identities in the absence of an Ahd-B+ gene in cis.

Much of the work we report is devoted to testing two models for how the inh5,6,7 region might function in

in85,6,7 Transvection

P

827

r " \ I ,. . I '.

b

, . ' ,

FIGURE 10.-Heterozygotes of T(2;3)Inu""' + Df(3R)R$4-8 with Df(3R)ullO ( a ) and R(ull0) (b-d) chromosomes. ( a ) A T(2;3) 62~""' + D f 3 R ) RS4-8/ Df3R)ullO heterozygote. Note that the phenotype of this animal is indistinguishable from that of the Df(3R)R$4-8/Df(3R)ullOheterozygote shown in Figure 4. ( b ) A 7'(2;3)b~u'~"~ + Df(3R)Rs4-8/ R(ullO)#lOheterozygote. Note very weak mottling of pigment in A6 and A7. When heterozygous with wild f~pe, R#lO [ =T(2;3)40;88B] showed pairing at 89E in 8 /40 salivary gland nuclei examined. ( c ) A T ( 2 ; 3 ) 6 ~ ' ~ ' " + Df(3R)Rs4-8/R(ullO)#l heterozygote. Note moderate mottling of pigment in A6 and A7. R#l is a complex T(2;3) with a break in 89D and showed pairing with wild type at 89E in 14 of 6.5 salivary nuclei examined. ( d ) A T(2;3)/ncr""' + Df3R)R$4-8/R(ul l0)#4 heterozygote. Note that pigmentation in posterior segments is suppressed to a greater extent than in the Df(3R)RS4-8/R(u110)#4 heterozygote (see Figure 9a) .

the absence of Abd-B+ in cis. Initially, our favored model was that iab-5,6,7could influence the expression of abd- A+ in cisin some way so as to assign A5 and A6 identities. Several lines of evidence suggesting that iab-5,6,7might have such an ability have already been reported ( KARCH et dl. 1985; DUNCW 1987; CELNIKER et al. 1990). How- ever, as shown in Figure 7, we established that the iab- 5,6,7region retains the ability to assign A5 and A6 identities when flanked by both an abd-A null allele (abd-

and an Abd-B deficiency ( Abd-B"") . Indeed, the iab-5,6,7 region retains function even in the triple mutants Ubx' abd-A"" Abd-B"" and Ubx"s'2 abd-A " A bd- B"', demonstrating that iab5,6,7 can promote A5 and

A6 identities in the absence of function in cis of all three genes in the BX-C known to encode transacting products. We note that our failure to detect any effect of an clbd-A null allele on iab-5,6,7 function does not rule out the possibility that abd-A' can promote A5 identity in certain circumstances. Indeed, we have obtained evidence, to be presented elsewhere, that abd- A' does have a weak dose-sensitive activity of this type.

Ultimately, the second model tested was found to be correct. The iab-5,6,7 region promotes A5 and A6 identities in the absence of Abd-B' in cis by the pairingdependent regulation of AM-B' in trans. Pairingde- pendent interactions were discovered in the Ubx do-

828 R. Hopmann, D. Duncan and I. Drlncan

FIGURE 11.-Evidence that distal inb7or the region between iab7and A6d-B is required for frnn.s-action of in85,6,7. ( a ) Male heterozygote of Tp(3;Y)iai+7"" and D/(3R)RS4-8. Tj1(3;Y)inl+7~'" is a transposition in which the region from 89B through in/+ 7 is inserted into the Y chromosome. The BX-C breakpoint is at about + 150 kb, located either between in67 and AM-B or within distal ia67. D/(3R)RW-8 is deficient for 89C through in67. Note that A6 and A7 show mottled pigmentation. This mottling is consistent with the deletion analysis presented in this report and presumably results from the action of the Y-borne inb5,6,7 region from Tj,(3;Y)ia67""on either the Ald-Bgene of Tp(3;Y)in/~7"~'or D/(3R) RY4-8, or both. Essentially the same phenotype is seen in T~~(3;Y)ia67s'"/D/(3R)P9heteroz)rgotes [ D/(3R)P9is deficient for the entire BX-C] . ( b ) Heterozygote of In(312R)inl+ 7"fsz and D/(3R)RS4-8. In(31.R)iab7"s2 is broken within the inl+/'region, between + 139.5 and + 142 kb. Males of this genotype show an almost complete lack of A5-type pigmentation in their posterior segments, indicating that the in6-5,6,7 region in In(31aR)ia/+7"r2 is unable to interact with AM-R in trans. The striking difference in phenotype as compared with a suggests that the region between the DX-C breakpoints of In(31,R)inb7"fSz and T1,(3;Y)ial+7S'" is essential for trans-interactions of ia65,6,7. In (3LR) iab7'fs2/ D/(3R)I'9 heterozygotes are virtdly identical in phenotype.

main of the BX-C by E. B. LEWIS ( 1954), who called the phenomenon transvection. Since then, transvection has been found at a number of loci in Drosophila (re- viewed by Wu 1993). Our initial tests of transvection would easily have detected fmn.Finteractions of the type and strength previously characterized in the Ubx domain but were uniformly negative. Several rearrangements induced on the Abd-B"'s and n/)d-A"24 A/)d-R"'* chromosomes, selected by their ability to disrupt transvection in the Ubx domain, have no effect on transvection at iaB5,6,7. Even a rearrangement broken within the BX-C [ Zn(3R)a/xl-A""] has no effect. It was only when we isolated rearrangements that cause very severe pairing disruptions in the vicinity of the BX-C that we saw significant impairment of the tmn+action of i a b 5,6,7. Such rearrangements were induced on Df(3R)ullO [ =Df(3R)89E;90A] that, by removing a region of homology distal to the BX-C, presumably in- tensifies the pairing disruption caused by rearrangements broken proximal to the complex. Two such rearrangements recovered, R(ullO)#4 [ = T (2;s) 40;89E; the break in 89E is within the bxd region of the BX-C] and R(ullO)#6 ( a complex rearrangement in which the 89E doublets, and little else, are transposed to a location between 61F and heterochromatin) cause near complete disruption of pairing of the BX-C in salivary gland chromosomes. However, even these rearrange-

ments only partially impair the tmwaction of iab5,6,7 when heterozygous with Df(3R) U~X". '~- ' . This result is particularly striking because Df(3R)ullO and Df(3R)- #lJ/,x Its4 - R share essentially no homologous sequences with which to pair over the large region from 89C through 90A. Twelve additional rearrangements induced on Df(3R) ull0 cause weaker variegated suppression of A5 and A6 identities.

The variegated pigmentation caused by many of the R ( u l l 0 ) rearrangements is patchy and resembles patterns produced by heterochromatic position effect variegation. However, it is very unlikely that classical position effect variegation is the cause of these patterns. Although the R#4 and R#6 rearrangements place the BX-C next to heterochromatin, others, including the moderately strong variegators R#l and R#13 (see Table 3 ) , do not. Moreover, we do not see inactivation of nbd- A + in R#4, contrary to what would be predicted from the "spreading" of heterochromatic position effect suppression from the R#4 breakpoint in the bxd region. Also, the enhancement of pigment loss in K#4/ Df(3R) UbxNS4-" heterozygotes by T ( 2 ; 3 ) b~'"''~ is consistent with disruption of transvection but is not predicted by the position effect variegation model. In fact, although many BX-C rearrangements broken in heterochromatin have been recovered, none have been found to cause spreading-type position effect variegation of

ia&5,6,7 Transvection 829

89B 9OB Df SI0

iab-5,6,7

VE 89D RGURE 12.-Schematic drawing showing synapsis of the over-

lapping deficiencies Df(3R) SI0 [ = Df(3R) 89B9-16;E3-4] and Df(3R)ullO [ = Df(3R) 89E3-4;9OA2-3]. Note the “bub- ble” of nonhomology in which synapsis is absent. As indicated by the shaded arrow, ia&5,6,7 is able to regulate AM-B by interaction across this region of asynapsis. The diagram is drawn approximately to scale. In molecular terms, the dimen- sions are large; the circumference of the asynapsed region is about 1 megabase ( HEINO et al. 1994).

BX-C genes (E. B. LEWIS, personal communication). Perhaps boundaries that function to define domains of condensed chromatin in the BX-C during normal development (PEIFER et al. 1987; GWRKOWCS 1990; P m o 1990; ORLANDO and P m o 1993) prevent the spread of heterochromatization within the complex.

Probably the most striking aspect of iab5,6,7transvec- tion is that it does not depend upon sequence homology or synapsis within the BX-C. This is demonstrated by the Df(3R)SIO/ Df(3R)ullO heterozygote, in which iab5,6,7 and Abd-B are apparently able to interact even though located on chromosomes that carry no BX-C sequences in common. Indeed, these chromosomes share no sequences from 89B through 90A, a distance of almost 1 megabase ( HEINO et al. 1994). Although synapsis in Df(3R) SIO/Df(3R) dl0 heterozygotes must be absent in this large region, pairing of flanking regions presumably brings iab5,6,7 and Abd-B into close proximity, facilitating their interaction (see Figure 12). Action of iab5,6,7 in trans is the only case of transvection known to be independent of homology in the target gene. The homology independence of iab5,6,7 transvection implies that the iab5,6,7 region can interact with its target in Abd-B over some distance. This is consistent with the normal function of iab5,6,7, which must interact over some 20-60 kb of intervening DNA to control Abd-B in cis in wild type. The nature of the interaction between iab5,6,7 and Abd-B in cis or trans is not known. Although a simple looping and contact mechanism seems more likely, mechanisms based on the short-range action of labile regulatory RNAs from iab5,6,7 (S~CHEZ-HERRERO and AKAM 1989; CUM- BERLEDGE et al. 1990) are also very attractive. transaction of iab5,6,7by the local titration of regulatory mole- cules is also possible.

It is perhaps worth pointing out that the lack of requirement for local synapsis does not disqualify trans action of iab5,6,7 as a transvection effect. In his intro-

duction of the term, LEWIS ( 1954) defined transvection as a “position effect revealed by modifylng the /rnns heterozygote by means of chromosomal rearrangements.” This definition subsumes any proximity-dependent transinteraction and is clearly consistent with our observations.

A second striking feature of iab5,6,7 transvection is the severity of pairing disruption required to achieve any phenotypic effect. Cases of transvection in the Ubx domain are vastly more sensitive to pairing disruption. As found by LEWIS ( 1954), the “critical region” within which breakpoints of simple rearrangements can cause strong suppression of transvection in the Ubx domain is very large, extending from the centromere (section 81 ) to the BX-C (89E). We cannot give a comparable estimate of the iab5,6,7critical region because the only condition in which we have seen disruption of the interaction is in deficiency heterozygotes in which homologues share essentially no homologous sequences in the vicinity of the BX-C. Even in this situation, only rearrangements broken very nearby have any strong effect. It is striking that none of the rearrangements isolated, including two broken within the BX-C itself, completely suppresses the ability of inb5,6,7 to promote A5 and A6 identities. This is probably because none totally prevents iab5,6,7 from interacting with Abd-E’ in trans. However, we cannot rule out the possibility that the pigment remaining in the most strongly suppressed genotype [ R#6/Df(3R) U~X‘‘~’~-’] results from nbd-A expression influenced by iab5,6,7 in cis.

Because disruption of inb5,6,7 transvection can be detected only in genotypes in which synapsis within the BX-C is totally absent to begin with, it is difficult to compare the strength of transinteraction with other cases of transvection. However, iab5,6,7 transvection is clearly far more difficult to disrupt than is transvection at Ubx (LEWIS 1954), d e ( GELBART 1982) and Scr ( PAT- TATUCCI and UUFMAN 1991). Whether tmnsinterac- tions between iab5,6,7 and Abd-B are more avid than those at w (GANS 1953; GREEN 1967; JACK and Junn 1979; SMOLIK-UTLAUT and GELBART 1987) or y ( GEYER et al. 1990) is not clear, because tests comparable to ours have not been made at these loci.

The ability of inb5,6,7to act over long distances likely provides an explanation for what appeared initially to be a paradox: How can two genes in the same complex have radically different pairing requirements for transvection? That is, how can the very same rearrangement cause almost complete suppression of transvection in the Ubx domain of the BX-C but have no effect upon iab5,6,7 transvection? The answer may be that in the Ubx domain transvection is a short-range interaction that requires synapsis within the complex, whereas iab- 5,6,7 transvection operates over greater distances and does not require local pairing. Thus, rearrangements that disrupt synapsis within the BX-C, but often leave


homologous complexes nearby in the nucleus, could disrupt Ubx but not iab5,6,7 transvection. A similar explanation has been put forward by SMOLIK-UTLAUT and GELBART (1987) to account for transvection at w. An alternate explanation is suggested by the variegated pigmentation seen when iab5,6,7transvection is disrupted. Pigmented patches are often well defined in these animals, suggesting these patches may be clonal, a possibility now being tested. If so, it could be that transient interaction between iab5,6,7 and Abd-B elicits some sta- ble change in Abd-B that is then inherited through cell division. In this case, the final phenotype would repre- sent the integration of all interactions occurring during development, so that even quite rare events would have a major phenotypic effect. Transvection at Ubx may be much more sensitive to pairing disruption because here effective interaction of sequences in trans requires con- tinual association. Finally, a third explanation could be that pairing near 89E is much more avid in the abdominal segments (in which iab5,6,7 is active) than in the third thoracic segment (in which Ubx transvection is typically monitored) .

In all of our genotypes in which iab5,6,7 acts solely in trans, A5 and A6 identities are shifted one segment posteriorly and occur in A6 and A7, respectively (see Figure 4) . Although the iab5 and iab6 regions are responsible for assigning A5 and A6 identities during normal development ( LEWIS 1978; DUNCAN 1987), it is not at all clear that these regions direct A5 and A6 identities when acting in trans. Indeed, it seems likely that A5 and A6 identities are determined by iab6 and iab7 in our deletion heterozygotes. The interactions of these regions with Abd-B in trans may well be impaired relative to their normal interactions in cis. This effect combined with the fact that our genotypes carry only one dose of Abd-B may result in Abd-B levels in A7 that are similar to those normally present in A6 and levels in A6 similar to those normally present in A5. Assignment of A5 identity in the absence of iab5 has already been demonstrated in other genotypes by CROSBY et al. (1993).

The properties of iab7 rearrangements indicate that the region between distal iab7 and Abd-B is required for transaction of iab5,6,7. We find that the iab5,6,7 region is unable to interact in trans in the i ~ b - 7 ~ ~ ’ inversion (broken in the interval +139.5-142) (KARCH et al. 1985) but retains this ability in Df(3R) ull0 (broken at +145.5-148.5). Thus, the right boundary of the region required for tranpaction of iab5,6,7 must lie between these breakpoints, in the region from +139.5 to +148.5 kb. The extent to which this transvection mdiat- ing region ( tmr) extends proximally in the BX-C is not known, nor is it clear that the tmr is distinct from iab7 itself. However, the observation that the ability of iab6 to act in trans is blocked by the i ~ b 7 ~ ~ ’ breakpoint demonstrates that the iab regions do not act on their own in trans and suggests they may share a distal site

that mediates their transinteractions with Abd-B. An ap- pealing possibility is that the tmr also mediates interactions with the Abd-Bpromoter in cis. If so, the tmrwould have to be located distal to iab7”, a deletion of the material from + 124.7 through +143 kb ( GALLONI et al. 1993). This deletion behaves as iab7- but does not impair the ability of iab6 to promote Abd-B expression in A6. Although the mechanism by which the tmr functions is unknown, an attractive possibility is that it facili- tates direct contact between the iab5,6,7 region and Abd-B. Alternatively, if transinteractions between iab 5,6,7and Abd-B are mediated by the iab R N A s , then the tmr could serve as a promoter for these RNAs.

It should be mentioned that several iab7 rearrangements, including some broken proximal to i ~ b 7 ~ ~ * , behave very differently from i ~ b - 7 ~ ” and show strong pigmentation of A5 and more posterior segments when hemizygous ( KARCH et al. 1985; GWRKOVICS et al. 1990). Some also show strong pigmentation of A4 and thus show more extensive pigmentation than wild type. The interpretation of these rearrangements is difficult, because many (perhaps all) are associated with a gain of function. As suggested by CELNIKER et al. ( 1990), the promotion of pigmentation by these rearrangements very likely results from altered abd-A expression. Al- though a completely satisfactory explanation for these effects has not been formulated, it is the absence of pigmentation in i ~ b - 7 ~ ~ ’ hemizygotes and in hemizygotes of at least some other inb7 rearrangements (SANCHEZ- HEREWRO et al. 1985a; DUNCAN 1987) that is of key importance here, because this forces the conclusion that iab5,6,7 does not interact with Abd-B in trans in these rearrangements.

Unlike many cases of transvection, transvection of iab5,6,7 appears insensitive to the allelic state of the reste gene. Most, but not all, cases of transvection at Ubx are totally suppressed by z“-type alleles ( KAuFMAN et al. 1973; BASU and BHAT 1981; GELBART and WU 1982; but see MART~NEZ-LABORDA et al. 1992). Transvection at dpp is suppressed by several types of allele, including rn ( GELBART and WU 1982) , whereas transvection at w (JACK and JUDD 1979) and y (GEER et ul. 1990) re- sponds only to specific alleles ( .zl and z”77h, respectively) . Transvection at Scr, like that at iab5,6,7, appears to be resteindependent (PATTATUCCI and

Transvection of the iab5,6,7 region may explain an unexpected property of our Ubx’ abd-ADZ4 Abd-B”” triple mutants and the triple of CASANOVA et al. ( 1987) . Both types of triple differ significantly in phenotype from a deficiency of the BX-C when heterozygous with wild type in adults. The triple mutants cause much weaker transformations of the posterior segments toward the anterior and are fertile in both sexes. Very likely the weakness of these effects is due to transvection of the iab regions. In support of this, we find that triple

KAUFMAN 1991 ) .


mutant/ deficiency heterozygotes carrying an insertion of 89D-E into the X heterochromatin [Dp(3;1)68], a genotype in which pairing of the BX-C is presumably very strongly disrupted, are similar in phenotype to + / deficiency heterozygotes. As an aside, we also draw at- tention here to our observation that embryos homozygous for Ubx’ abd-ADZ4 Abd-BD’* triple mutants are indistinguishable in cuticular structures from embryos homozygous for deficiencies that remove the entire BX- C. This strongly supports the conclusion that all segment identity functions of the BX-C are executed by the products of Ubx, abd-A and Abd-B. Although the triple mutant of CASANOVA et al. (1987) is commonly (and quite correctly) cited in this regard, their triple was generated by repeated rounds of mutagenesis and the very real possibility of damage outside of Ubx, abd- A and Abd-B was not ruled out. The probability of such damage in our triples is very low, because these were generated by recombination.

Transvection possibly similar to that at iab5,6,7 has recently been described for the iab3 and iab4 regions by JIJAKLI and GHYSEN ( 1992) . These authors report evidence that the iab3 and iab4 regions can act in trans on abd-A+ to suppress the development of “lateral dot” structures in the larval central nervous system in posterior abdominal segments. Although the pairing requirements of this case of transvection were not characterized in any detail, these authors do present evidence that iab3,4 transvection is not totally disrupted in heterozygotes for Tp(3;1)P115, a transposition in which the region from 89B through 89E is inserted into the X heterochromatin ( LINDSLEY and ZIMM 1992). This observation suggests that iab3,4 transvection may be as difficult to disrupt as transvection at iab5,6,7. In work to be presented elsewhere, we find (in Mcp Abd-B”’* heterozygotes) that the iab5,6,7 region can interact with Dp(3;1)P115 in trans to a substantial degree.

The extreme pairing disruption required to impair transvection of the iab5,6,7 region rendered the phenomenon rather difficult to demonstrate. This raises the possibility that similar cases of transvection exist at other loci but have gone unrecognized. One good candidate is engruiled (en) . KORNBERG ( 1981 ) found that cytologically normal lethal alleles partially comple- ment en’, whereas lethal alleles associated with rearrangement breakpoints at en do not. To test whether the partial complementation of unrearranged alleles is due to transvection, GUSTAVSON and KORNBERG (personal communication) irradiated a cytologically normal lethal allele and selected derivatives in which complementation with en’ was suppressed. Apart from one en- hancer mutation, the only rearrangements recovered were broken at the en locus itself. A probable explanation is that partial complementation of en’ involves very strong transinteractions of the type we describe at iab 5,6,7. Like iab5,6,7 transvection, partial complementa-

tion of en’ by lethal alleles is not zeste dependent (CON- DIE and BROWER 1989) . The possibility of transvection at en is much strengthened by the recent work of KASSIS (1994) and KASSIS et al. (1991), who analyzed germ- line transformants carrying fragments of en DNA. They find several transformants in which the homozygote, but not the heterozygote, shows repression of the vec- tor-derived white gene. This repression appears to be pairing dependent, as it is not seen when fragments inserted at widely different sites are combined. Like iab 5,6,7 transvection, the interaction of these en “pairing sensitive” sites is zste independent and appears to be long range, in that moderately distant elements appear to interact. Long-range interaction is also indicated by the “homing” of these elements to en and other loci during P-element-mediated transformation ( HAMA et al. 1990; KASSIS et al. 1991; KASSIS 1994). It is likely significant that the distal iab7 region, which we suggest may play an important role in iab5,6,7 transvection, also appears to cause homing of a Pelement construct (GAL LONI et al. 1993). Recently, pairing-sensitive elements have been found at several loci [ FAWARQUE and DURA 1993; see unpublished examples cited in KASSIS (1994) 1 , suggesting that transvection of the iab5,6,7 type may be rather common.

Finally, our results suggest that transvection may be more widespread and of far greater general significance than usually imagined. We demonstrate that transvection at iab5,6,7 is proximity dependent but does not require synapsis within, or for some distance to either side of, the BX-C. Even in the absence of local synapsis the transinteraction is extraordinarily tenacious. There would seem to be no reason why similar synapsis-independent interactions could not occur in organisms that lack somatic pairing altogether. transinteractions could be facilitated in such organisms solely by the proximity of alleles within the nucleus. The idea that somatic pairing is not an absolute requirement for trans4nteractions between alleles was first put forward by E. B. LEWIS (1954) in his seminal paper on transvection. Clearly stated in this paper is the view that interactions between alleles are “a function of the distance separating homologous chromosomes” and that somatic pairing provides only a “greater possibility” of interaction than is present without pairing. Very likely, transvection of the iab 5,6,7-type is responsible for paramutation in plants (BRINK 1973; MEYER et al. 1993; PATTERSON et al. 1993) and may also be involved in certain human disorders [see e.g. the discussion of TARTOF and HENIKOFF (1991)l.

We thank ED LEWIS for many discussions and for providing many of the stocks used in this study, ELIZABETH GUSTAVSON and TOM KORNBERG for allowing us to cite unpublished observations and MARK BIGGIN, KEN BURTIS, BURKE JUDD, CINES MORATA and DAVID KUHN for providing stocks. SHICE SAKONJU and DAVID HOGNESS generously provided BX-C phage clones. We thank the anonymous reviewers for


their constructive comments. Special thanks go to PAULA KIEFEL for help in mapping breakpoints, RAGHU SINCH and JOYCE YEE for isolating many of the Mc revertants and PAT O'FARRELL for pushing us to look harder for transvection. This work was supported by a grant from the National Institutes of Health.

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