trans-splicing of the mod(mdg4) complex locus is ...the modiﬁer of mdg4, mod(mdg4), locus in...

Copyright © 2005 by the Genetics Society of AmericaDOI: 10.1534/genetics.103.020842

Trans-splicing of the mod(mdg4) Complex Locus Is Conserved Between theDistantly Related Species Drosophila melanogaster and D. virilis

Manuela Gabler,* Michael Volkmar,* Susan Weinlich,* Andreas Herbst,*Philine Dobberthien,* Stefanie Sklarss,* Laura Fanti,† Sergio Pimpinelli,†

Horst Kress,‡ Gunter Reuter* and Rainer Dorn*,1

*Institute of Genetics, Martin Luther University, D-06120 Halle, Germany, †Department of Genetics and Molecular Biology, University LaSapienza, I-00185 Rome, Italy and ‡Institute for Biology-Genetics, Free University of Berlin, D-14195 Berlin, Germany

Manuscript received August 4, 2003Accepted for publication October 29, 2004

ABSTRACTThe modifier of mdg4, mod(mdg4), locus in Drosophila melanogaster represents a new type of complex gene

in which functional diversity is resolved by mRNA trans-splicing. A protein family of �30 transcriptionalregulators, which are supposed to be involved in higher-order chromatin structure, is encoded by bothDNA strands of this locus. Mutations in mod(mdg4) have been identified independently in a numberof genetic screens involving position-effect variegation, modulation of chromatin insulators, apoptosis,pathfinding of nerve cells, and chromosome pairing, indicating pleiotropic effects. The unusual genestructure and mRNA trans-splicing are evolutionary conserved in the distantly related species Drosophilavirilis. Chimeric mod(mdg4) transcripts encoded from nonhomologous chromosomes containing the splicedonor from D. virilis and the acceptor from D. melanogaster are produced in transgenic flies. We demon-strate that a significant amount of protein can be produced from these chimeric mRNAs. The evolutionaryand functional conservation of mod(mdg4) and mRNA trans-splicing in both Drosophila species is further-more demonstrated by the ability of D. virilis mod(mdg4) transgenes to rescue recessive lethality of mod(mdg4)mutant alleles in D. melanogaster.

THE majority of genes in higher eukaryotes repre- encoded by the four 5�-exons but differ in their C-termi-nal region encoded by alternative 3�-exons. This kindsents monocistronic units where noncoding intron

regions interrupt the protein-coding exon sequences. of trans-splicing clearly differs from splice leader trans-splicing that predominates in Caenorhabditis and Try-The resulting mature mRNA usually encodes a uniquepanosomes where polycistronic transcripts are resolvedpolypeptide. Recent advances in genome analysis of sev-by addition of noncoding leader sequences (Blumen-eral model organisms and the molecular characteriza-thal 1998). Mutational dissection and differential bind-tion of a large number of genes revealed that alternativeing of Mod(mdg4) isoforms on polytene chromosomespre-mRNA splicing is one of the main mechanisms gen-suggest that the variable C-terminal regions encoded byerating a highly expanded proteome diversity. Thus,any of the alternative 3�-exons determine functionalprotein families with slightly different isoforms or evenspecificity. Specific Mod(mdg4) isoforms are supposedproteins with unrelated functions can be produced fromto be involved in control of heterochromatic gene si-single or multiple promoter elements within one genelencing, regulation of homeotic genes, function of chro-(for review, see Graveley 2001; Maniatis and Tasicmatin insulators, nerve cell pathfinding, induction of2002). Regulatory integration of different transcriptionalapoptosis, and control of meiotic processes (Dorn etunits is found in gene complexes like Hox genes, hemo-al. 1993; Gerasimova et al. 1995; Harvey et al. 1997;globin genes, or immunoglobin genes. This organiza-Gorczyca et al. 1999; Buchner et al. 2000). Genomiction reflects clustering of genes with related functions.structure and transgene analysis demonstrate the spe-With mod(mdg4) a new type of functional clustering hascific functional organization of the complex mod(mdg4)been discovered in Drosophila. This complex locus en-locus (Dorn et al. 2001). Mature mod(mdg4) transcriptscodes �30 isoforms generated by mRNA trans-splicingare generated by a trans-splicing mechanism combining(Buchner et al. 2000; Dorn et al. 2001; Krauss andone primary transcript comprising the common fourDorn 2004). Protein isoforms produced by mod(mdg4)5�-exons with another transcription unit contributingcontain a common 402-amino-acid N-terminal regionone of the alternative 3�-exons. A comparably complexgene structure was also described for a number of othergenes in Drosophila, including Broad, tramtrack, GAGA-

1Corresponding author: Institute of Genetics, Martin Luther Univer-factor/Trl, and lola, all of which encode numerous pro-sity, Weinbergweg 10, D-06120 Halle, Germany.

E-mail: [email protected] tein isoforms with alternative C termini (DiBello et al.

Genetics 169: 723–736 (February 2005)

724 M. Gabler et al.

indicate the chromosomal location of the transgene. The ge-1991; Read and Manley 1992; Soeller et al. 1993; Mad-nomic 6.8-kb Not I-XbaI fragment exclusively containing theden et al. 1999). Interestingly, in addition to mod(mdg4),common exons 1–4 was cloned into pW8 and one second

mRNA trans-splicing was recently reported for the lola chromosomal transgenic semilethal line was established.locus (Horiuchi et al. 2003). Another unique character- Transformation of D. melanogaster was performed as described

by Rubin and Spradling (1982).istic of these genes is that they all encode BTB/POZThe chromosomes 3-P(w� Dv mod(mdg4) 11.5kb Not I) moddomain proteins, which frequently contain Cys2His2

(mdg4)neo129 and 3-P(w� Dv mod(mdg4) 11.5kb Not I) mod(mdg4)02zinc-finger motifs within the variable C-terminal region.

were obtained by recombination. The presence of both theTherefore, mod(mdg4) appears to represent a proto- transgene and the mod(mdg4) mutations was tested for with

type of a new class of complex loci where functional specific PCR primer pairs. Recombinant chromosomes, whichhad lost the w� marked transgene, did not complement thevariety is produced by a trans-splicing mechanism com-original mod(mdg4) mutations.bining independent transcription units in a regulated

Drosophila strains and crosses: mod(mdg4) mutant alleles aremanner.described in Buchner et al. (2000). The allele mod(mdg4)07/351

In working toward a better understanding of the was obtained from M. Frasch. Other strains are described inmod(mdg4) complex structure and the underlying con- Lindsley and Zimm (1992).

All crosses were performed at 25�. For complementationserved principles involved in mRNA trans-splicing, weanalysis mod(mdg4) mutant strains containing the w m4 chromo-analyzed the Drosophila virilis ortholog of mod(mdg4).some and the TM3, Sb Ser balancer have been used. One copyMolecular analysis of D. virilis mod(mdg4) revealed strongof the D. virilis mod(mdg4) transgene was inherited maternally

conservation of gene structure and the encoded protein and its presence in offspring flies was monitored by the w�

isoforms. Functional conservation is suggested by simi- marker gene. The percentage of homozygous/trans-heterozy-gous mod(mdg4) mutant flies was calculated as a percentagelar protein distribution of ortholog isoforms on poly-of the expected number of these flies (only flies containing thetene chromosomes in D. virilis and Drosophila melanogas-transgene were included in this calculation) in the appropriateter and by mutant rescue in transgenic D. melanogastercrosses. All mutant alleles do not complement in the absence

lines after expression of D. virilis mod(mdg4) protein of the transgenes under the conditions used. This is confirmedisoforms. Moreover, we show that mRNA trans-splicing by the absence of homozygous/trans-heterozygous mod(mdg4)is conserved in both species, allowing the generation of mutant flies without the D. virilis transgene in all crosses.

RNA isolation and RT-PCR: Isolation of poly(A)� RNA waschimeric D. melanogaster/D. virilis mod(mdg4) transcriptsperformed as described in Dorn et al. (2001). Primers usedin vivo. Quantitative RT-PCR experiments reveal thatin RT-PCR experiments are ex4-virF, 5�-CGAGCACCGCCAACmod(mdg4)-67.2 chimeric transcripts consisting of a do- GTAATTGATC-3�; 64.2-B-RT, 5�-CAA/gCTTGCAG/cTCCT

nor encoded by a third chromosomal transgene and an TGCCG/aTC-3� (different nucleotide positions in D. virilis areacceptor encoded by the third chromosomal endoge- indicated by small letters); 51.4-B-RT, 5�-CAAGACCAATAAG

TTTTCAATCCCG-3�; and 56.3-B-RT, 5�-ACATCGCCGCTCCnous locus represent �12% with respect to the endoge-TGGTCC-3�.nous D. melanogaster mod(mdg4)-67.2 transcript. The cor-

The new isoform mod(mdg4)-53.5 was identified with primersresponding chimeric protein can be detected at nearly ex4-melF, 5�-CGCAAATGTTATGGACCCTCTC-3� and 53.5-wild-type level on polytene chromosomes of transgenic RT-Bmel, 5�-CGGCTTGTGATTGTGAAATCCTC-3� in D. mela-mod(mdg4)� homozygous larvae. Our data provide new nogaster and primers ex4-virF, 5�-CGAGCACCGCCAACGT

AATTGATC-3� and 53.5-RT-Bvir, 5�-GTAATCCTGATGCTTGinsight into functional conservation of a new type ofTGGAGC-3� in D. virilis. Total RNA was isolated with TRIzolcomplex loci in which functional complexity is resolved(Invitrogen, San Diego) and poly(A)� RNA was obtained withby mRNA trans-splicing.the mRNA purification kit (Amersham, Buckinghamshire,UK). For reverse transcription (RT), 1 �g RNA was incubatedwith random hexamer primer and Moloney murine leukemiaMATERIALS AND METHODSvirus reverse transcriptase (Promega, Madison, WI) accordingto the manufacturer’s protocol. PCR reaction mixture con-Library screen: Screening of a D. virilis genomic librarytained 167 �m dNTPs, 1.67 mm MgCl2, 267 nm of each primer,(Lanio et al. 1994) was performed as described in Sambrookand 1.5 units of Taq DNA polymerase. Conditions for PCRet al. (1989). Washes were performed twice in 2� sodiumwere 95� for 5 min, 95� for 40 sec, 55� for 40 sec, and 72� forsodium citrate and 0.1% sodium dodecyl sulfate for 10 min.40 sec (35 cycles).Two cDNA clones corresponding to isoforms mod(mdg4)-67.2

Real-time quantitative RT-PCR: Total RNA was extractedand mod(mdg4)-58.8 were used as radiolabeled hybridizationfrom adult female flies of strains w 1118, w 1118 3-P(w� Dvprobes. Three recombinant genomic �-clones were isolatedmod(mdg4) 11.5kb Not I)/3-P(w� Dv mod(mdg4) 11.5kb Not I), andand the genomic insert of the representative clone �Dv2-1 wasw1118 3-P(w� Dv mod(mdg4) 6.8kb NotI-XbaI)/3-P(w� Dv mod(mdg4)sequenced and used in all other experiments. The overlapping6.8kb Not I-XbaI) using the TRIzol reagent (Invitrogen). PCRrecombinant �-clone Dv3-2 was isolated in a second libraryprimers were selected for intron spanning using the GeneRun-screen with the help of a probe deduced from �Dv2-1.ner software (Hastings Software). The primer sequences areThe GenBank/EMBL accession number of the D. virilisrp49-fwd, 5�-TGTCCTTCCAGCTTCAAGATGACCATC-3�;mod(mdg4) genomic sequence is AJ586737.rp49-rev, 5�-CTTGGGCTTGCGCCATTTGTG-3�; mel-com-Construction of transgenic lines: The 11.5-kb genomic in-fwd, 5�-TTCTTCCGCAAGATGTTCACTCAGATG-3�; mel-com-sert of clone �Dv2-1 was cloned into the unique Not I site ofrev, 5�-TGAATTGGATGAGGTCCTTCAGCG-3�; vir-com-fwd,the P-transformation vector pW8 (Klementz et al. 1986). After5�-CGCACCGTTTGGTGTTGTCTGTCTGC-3�; vir-com-rev,transformation, three independent transgenic lines (chromo-5�-CTTATCAGGTCCTTCAATGCCGAATGGC-3�; mel-spec-somes 1, 2, and 3) were obtained. The transgene was desig-

nated as P(w� Dv mod(mdg4) 11.5kb Not I) ; Arabic numbers fwd, 5�-CAAATACGAGCGGTGGCGGAGTGAC-3�; vir-spec-fwd,

725Evolutionary Conservation of Trans-splicing

5�-ACACAAGCACCACCAGCGTCCAAGC-3�; mel-64.2-rev, producibly stained sites on D. melanogaster polytene5�-TGGCTGCGAATGAAACTGATCTCCG-3�; vir-64.2-rev, chromosomes (Buchner et al. 2000). A comparably low5�-GTTGTCTGGCCATTCGCTTGGGTC-3�; and mel-67.2-

number of stained sites is detected in polytene chromo-rev, 5�-TTTCGGTCTGCCGCGTTTACGTGG-3�. Specificity ofsomes of D. virilis (Figure 1A). However, the decreasedthe primers was confirmed by melting curve analysis, agarose

gel electrophoresis, and sequencing of the PCR products. The number of stained sites in D. virilis may be due to areal-time qRT-PCR was performed with Quantitect SYBR differential distribution and/or to lower affinity of theGreen RT-PCR kit (QIAGEN, Chatsworth, CA) on an iCycler antibody in D. virilis. Therefore we tested two individualThermocycler (Bio-Rad, Richmond, CA) in triplicate for each

binding sites, which are known to comprise relatedsample and analyzed with the iCycler iQ software (Bio-Rad).genes in the two species. Subdivision 33A in D. virilisFor data standardization, the absolute expression level of each

mRNA was determined and set in proportion to D. melanogaster harbors the gene vE74, the ortholog of the D. melanogas-rp49, which is strongly expressed in females (Tamate et al. ter E74A gene (Jones and Dalton 1991) corresponding1990). to the anti-Mod(mdg4)-58.0403-534 binding site 74EF. A

Immunological analysis: Staining of polytene chromosomessecond corresponding site is subdivision 58E in D. virilis,was performed as described in Buchner et al. (2000). Immu-which carries one of the three clusters of 5S rRNA genesnostaining was performed with anti-Mod(mdg4)-58.0BTB-534

(1:1000 dilution), which recognizes all Mod(mdg4) isoforms, (Kress 2001). In D. melanogaster, the 5S RNA genes areand with anti-Mod(mdg4)-58.0403-534 (1:100 dilution), which clustered in subdivision 56E (Wimber and Steffensonexclusively detects the isoform Mod(mdg4)-58.0. The antibody 1970). Both are strong binding sites for isoform Modanti-Mod(mdg4)-67.2403-610 was generously provided by P. Geyer

(mdg4)-58.0. Another example of a common binding(Iowa State University) and used in a 1:1000 dilution on polytenesite of Mod(mdg4) is the Bithorax-Complex (BX-C). Thechromosomes. The presence of the D. virilis transgene was

probed by PCR with a D. virilis-specific primer pair (primer D.vir observed homeotic transformation of the mutant allele7211F, 5�-TGATGTAAGTTGGGTTCCATTGCG-3� and primer mod(mdg4)neo129 indicated an involvement of mod(mdg4)D.vir 64.2B, 5�-GGATCCATGCAGCTTAAGCTTGTGCGA-3�), in the transcriptional regulation of the BX-C (Dorn etusing DNA isolated from the corresponding carcasses as tem-

al. 1993). To prove if Mod(mdg4) binds to the BX-C ofplate. Immunofluorescence and sequential in situ hybridizationboth species, we performed sequential staining withwith salivary gland chromosomes was performed as described in

Pimpinelli et al. (2000). anti-Mod(mdg4)-58.0BTB-534 and a probe of D. melanogas-Western analysis: Salivary glands of w1118, 2-P(w� Dv mod(mdg4) ter Ubx DNA on polytene chromosomes. In fact, anti-

11.5kb NotI)/�, mod(mdg4)02/mod(mdg4)02; mod(mdg4)02/mod Mod(mdg4)-58.0BTB-534 binds close to the Ubx probe in(mdg4)02, and 2-P(w� Dv mod(mdg4) 6.8kb NotI-XbaI)/�; mod

both species (Figure 1B), indicating its binding to ho-(mdg4)02/mod(mdg4)02 were dissected from third instar larvae inmologous sites. These results clearly point to the struc-IP buffer (20 mm Tris/HCl pH 8.0; 150 mm NaCl; 10 mm

EDTA; 1 mm EGTA; 2 mm Na2VO4). Equal protein amounts tural and functional conservation of the mod(mdg4) locuswere loaded and Western blots were probed with polyclonal in D. virilis.anti-Mod(mdg4)-67.2403-610 antibody (1:1000) and monoclonal For isolating the D. virilis mod(mdg4) locus, we selectedantitubulin antibody (1:20,000; Sigma, St. Louis).

two cDNA clones corresponding to isoforms mod(mdg4)-67.2 and mod(mdg4)-58.8 as hybridization probes andscreened a genomic D. virilis � library. Three indepen-RESULTSdent recombinant �-clones were isolated and restricted,

The genomic structure of the mod(mdg4) locus in D. among other endonucleases, with SalI. All recombinantmelanogaster and D. virilis is highly conserved: Recently, clones contained a 0.5-kb SalI restriction fragment, whichwe described the genomic structure of the mod(mdg4) is also present in the common exon 4 of D. melanogasterlocus in D. melanogaster. The structural organization of mod(mdg4). Sequence analysis of the cloned fragmentthis complex locus is unusual because both DNA strands revealed a significant sequence conservation within thisare used as coding strands and independent transcripts coding region. Subsequently, we sequenced the geno-are combined via mRNA trans-splicing (Dorn et al. 2001; mic insert of one representative �-clone. ComparisonLabrador et al. 2001). To prove if mod(mdg4) is con- with the corresponding sequence of D. melanogasterserved in distantly related Drosophila species, we tested mod(mdg4) revealed a strong conservation of both geno-the polyclonal antiserum anti-Mod(mdg4)-58.0BTB-534 for mic structure and Mod(mdg4) isoforms (Figure 2).crossreactivity in D. virilis. This antibody stains a number Most importantly, also in D. virilis the orthologous iso-of several hundred bands in salivary gland polytene forms Mod(mdg4)-53.1, Mod(mdg4)-62.3, Mod(mdg4)-chromosomes of D. melanogaster (Buchner et al. 2000). 55.6, Mod(mdg4)-53.6, Mod(mdg4)-54.7, Mod(mdg4)-A similar effect was obtained if polytene chromosomes 59.0, and Mod(mdg4)-67.2 are encoded by the oppositeof D. virilis were stained with the same antiserum (data DNA strand. These data strongly imply that mRNA trans-not shown). To prove the conservation of individual splicing is also conserved in both species. To verify ifMod(mdg4) isoforms and their chromosomal binding, the putative coding regions are transcribed in D. virilis,we stained D. virilis polytene chromosomes with the we performed RT-PCR experiments for six selectedspecific antibody anti-Mod(mdg4)-58.0403-534, detecting mod(mdg4) isoforms, mod(mdg4)-64.2, -60.1, -55.1, -53.1,exclusively the isoform Mod(mdg4)-58.0. This isoform -58.0, and -67.2, respectively. The forward primer corre-

sponds to the putative common exon 4 and the back-is significantly less abundant and restricted to �25 re-


Figure 1.—Distribution of Mod(mdg4) protein isoforms on sali-vary gland polytene chromosomesof D. virilis. (A) The polyclonal an-tiserum anti-Mod(mdg4)-58.0403-534

specifically recognizing the Mod(mdg4)-58.0 protein isoform in D.melanogaster shows a similar stain-ing pattern in D. virilis. (B) Local-ization of Mod(mdg4) at the BX-Cin D. melanogaster and D. virilis bysequential in situ hybridizationwith a Ubx-DNA probe and anti-body staining with anti-Mod(mdg4)-58.0BTB-534. In both species thebinding of Mod(mdg4) is closeto Ubx.

ward primers are isoform-specific primers hybridizing and variable, isoform-specific C termini encoded by al-ternatively spliced exon 5 or exons 5 and 6 (Buchnerdownstream of the putative open reading frames (ORF).

The resulting fragments were sequenced and the pre- et al. 2000). Comparison of the common region (Figure3) reveals a strong conservation (82% identity). Thedicted ORFs and the alternative splice sites were verified.

The exon/intron structure of the common 5�-region N-terminal BTB/POZ domain is identical in both spe-cies. Within the remaining common protein part (aminohas been determined by RT-PCR with a forward primer

deduced from the first coding exon and a backward acids 121–413) only a small number of amino acid re-placements (in most cases conservative ones) are pres-primer deduced from exon 4.

Comparison of the D. melanogaster and D. virilis Mod ent. The difference in size (413 vs. 402 amino acids) ismainly attributed to additional glutamine residues in(mdg4) protein isoforms: The multiple D. melanogaster

Mod(mdg4) protein isoforms contain a common N-termi- D. virilis.Next we compared the specific C termini of Modnal region of 402 amino acids encoded by exons 2–4


Figure 2.—Comparison of the genomic structure of mod(mdg4) of D. melanogaster and D. virilis. Above the scale the mod(mdg4)exon/intron structure of D. melanogaster and D. virilis is shown. Translated regions are represented as rectangles (solid, encodedby the same strand as common exons; shaded, encoded by the complementary strand). Nontranslated regions are shown as bars.The alternatively spliced specific exons and their corresponding orthologs in D. virilis are indicated by numbers representingthe deduced molecular weight of the D. melanogaster isoforms. The alternatively used splice site of exon 4 is indicated by multiplebars. Primers used in RT-PCR experiments are indicated by arrows. Genomic fragments tested for mutant rescue are indicatedbelow the scale.

(mdg4) isoforms. Figure 3 also shows the alignment of onic stages, we performed antibody staining with anti-Mod(mdg4)-58.0BTB-534, which detects all Mod(mdg4)specific C-terminal regions of five Mod(mdg4) isoforms.

Despite the strongly conserved amino acid positions of protein isoforms. In both species, Mod(mdg4) proteinscould be detected during early cleavage cycles (data notthe FLYWCH consensus sequence, most of the amino

acid positions within this motif are also conserved be- shown).D. virilis mod(mdg4) transgenes rescue mutant pheno-tween orthologous isoforms. In the case of isoform Mod

(mdg4)-67.2, the identity is 85% and isoforms Mod types in D. melanogaster : The strong structural conserva-tion of mod(mdg4) in D. virilis prompted us to test its(mdg4)-64.2 and Mod(mdg4)-60.1 show an identity of

90% within the FLYWCH motif. On the basis of this functional conservation. Therefore, we established trans-genic lines containing the genomic insert of the recom-observation we theorize that these amino acids contrib-

ute mainly to specific functions of the different Mod binant phage �Dv2-1, which encodes the D. virilis mod(mdg4) common exons, five specific exons from isoforms(mdg4) isoforms. Outside the FLYWCH domain the

sequence of the orthologous isoforms is less, but still mod(mdg4)-64.2, mod(mdg4)-60.1, mod(mdg4)-53.5, mod(mdg4)-55.1, mod(mdg4)-53.1, and a partial specific exonconserved. The isoforms Mod(mdg4)-55.1 and Mod

(mdg4)-58.0 do not contain the FLYWCH consensus from mod(mdg4)-62.3 (cf. Figure 2). This 11.5-kb geno-mic NotI fragment is expected to produce these five D.sequence. However, also in these cases the specific C

termini are significantly conserved (45 and 51% iden- virilis Mod(mdg4) protein isoforms, but not the Mod(mdg4)-62.3 because both the trans-splice site and atity, respectively). These results support our view that

the different orthologous Mod(mdg4) protein isoforms putative promoter driving its expression are not in-cluded. This prediction was confirmed by RT-PCR ex-have individual functions, which are conserved in both

Drosophila species. A comprehensive evolutionary anal- periments with two independent transgenic lines. Toprove the capability of the D. virilis P(w� Dv mod(mdg4)ysis of all Mod(mdg4) protein isoforms identified in D.

virilis and other Diptera species is presented in Krauss 11.5kb NotI) transgene to rescue the recessive lethalityof mod(mdg4) mutant alleles, strains containing the D.and Dorn (2004).

mod(mdg4) is expressed in all stages of development virilis transgene and one of the two recessive lethal al-leles, mod(mdg4)02 and mod(mdg4)neo129, have been con-and is maternally provided in both Drosophila species:

To compare the developmental expression pattern of structed. These strains have been used in complementa-tion crosses with mutant alleles mod(mdg4)neo129, modmod(mdg4) in both Drosophila species, we performed

Northern blot analyses. In a previous analysis with (mdg4)R32, mod(mdg4)02, and mod(mdg4)07. All alleles repre-sent mutations within the mod(mdg4) common regionpoly(A)� RNA, we detected abundant transcripts at 2.0

and 2.3 kb in all stages of development (Buchner et al. and do not complement each other in the absence ofthe transgene (Buchner et al. 2000; this work). The2000). As expected, D. virilis mod(mdg4) shows a very

similar expression pattern, characterized by abundant P(w� Dv mod(mdg4) 11.5kb NotI) transgene is able torescue almost all trans combinations of mod(mdg4) mu-transcripts during embryonic development and in fe-

males (data not shown). To prove if maternal Mod tant alleles used, independently of whether the trans-gene is located on the second or the third chromosome(mdg4) proteins are present in preblastoderm embry-


(Table 1). However, mod(mdg4)02 homozygotes are more rescue ability of the transgene is allele dependent. Ahigher number of trans-heterozygous offspring containingcompletely rescued by the third chromosomal transgene

(79.0%) as compared to the second chromosomal one the mutant allele mod(mdg4)neo129 as compared to trans-het-(26.6%, Table 1, columns 1 and 2). This may be due to erozygotes containing the mutant allele mod(mdg4)02 waschromosomal position effects. On the other hand, the rescued (Table 1). The latter allele is supposed to be a

loss-of-function allele, whereas mod(mdg4)neo129 is a hypo-morphic allele (Buchner et al. 2000).

However, the mutant allele mod(mdg4)07 behaves dif-ferently. Recessive lethality of trans-heterozygous P(w�

Dv mod(mdg4) 11.5kb NotI) mod(mdg4)neo129/mod(mdg4)07

offspring flies is not rescued (0.6% of expected flieshatch) and only a small number of mod(mdg4)02/mod(mdg4)07 offspring is rescued in the presence of the sec-ond or the third chromosomal P(w� Dv mod(mdg4)11.5kb NotI) transgene, 12.5 and 5.4%, respectively. Incontrast to most other recessive lethal mod(mdg4) alleles,the mutant allele mod(mdg4)07 is embryonic lethal andwas shown to contain substitutions of two conservedamino acids, which are expected to be involved in dimer-ization, within the BTB domain (D35N and G93S)(Read et al. 2000). This might indicate an antimorphicnature of this mutant allele.

We did not find significant differences, whether onecopy of the transgene is inherited maternally (Table 1)or paternally (data not shown). However, if two copiesof the second chromosomal transgene are inherited,one maternal and one paternal, homozygous mod(mdg4)02

flies are completely rescued (106.7% vs. 26.6% in thecase of one maternal copy). This indicates dosage-dependent effects of the transgene. We also did notobserve significant differences in rescue ability of femaleand male offspring, except in crosses with mutant allelemod(mdg4)R32. In these crosses, the number of trans -het-erozygous female offspring is significantly reduced com-pared to offspring males.

To determine if mutant rescue depends solely on theexpression of the transgene-encoded D. virilis mod(mdg4)isoforms or if interspecies mRNA trans-splicing is in-volved, we constructed the P(w� Dv mod(mdg4) 6.8kbNotI-XbaI) D. virilis transgene. This transgene exclusivelyencodes the common exons 1–4. Therefore, functionalMod(mdg4) proteins can be produced only via trans-

Figure 3.—Sequence comparison of orthologous Mod(mdg4) protein isoforms of D. melanogaster and D. virilis. (A)Alignment of the common N-terminal protein sequences en-coded by exons 2–4 from D. melanogaster and D. virilismod(mdg4). The BTB domain is boxed (dark shading). (B)Comparison of the putative specific C-terminal protein se-quences of the isoforms Mod(mdg4)-64.2, Mod(mdg4)-60.1,Mod(mdg4)-67.2, Mod(mdg4)-55.1, and Mod(mdg4)-58.0.Identical amino acid positions are indicated by asterisks; dou-ble dots and single dots indicate functional and structuralsimilar amino acids, respectively. The FLYWCH motif is indi-cated by a box (light shading) and the strongly conservedamino acid positions within the FLYWCH motif are in boldfacetype.


splicing. Complementation analysis with a second chro-mosomal transgene revealed that this transgene alsopartially rescues recessive lethality (Table 1). This resultsuggests that proteins encoded from chimeric tran-scripts facilitate mutant rescue. However, the numberof homozygous/trans-heterozygous offspring flies is sig-nificantly reduced compared to the appropriate geno-types containing the P(w� Dv mod(mdg4) 11.5kb NotI)transgene. Also fertility of female offspring is signifi-cantly reduced in the presence of the P(w� Dv mod(mdg4)6.8kb NotI-XbaI) D. virilis transgene in contrast to theappropriate females containing the longer transgene.

Chimeric mod(mdg4) transcripts are generated bymRNA trans-splicing of D. melanogaster and D. virilis pre-mRNAs: Recently, we have shown that trans-splicing ofmod(mdg4) pre-mRNAs is not restricted to the isoformsencoded by opposite DNA strands but also includesisoforms expected to be produced exclusively by cis-splicing (Dorn et al. 2001). This suggests that trans-splicing should be the general mechanism to produceall mod(mdg4) transcripts. Additionally, the rescue abilityof the D. virilis P(w� Dv mod(mdg4) 6.8kb NotI-XbaI) trans-gene indicates the generation of functional chimericmod(mdg4) transcripts.

To experimentally prove the existence of these tran-scripts, we initially performed RT-PCR, which allowedus to differentiate between the chimeric and the corre-sponding D. virilis transcripts (schematically shown inFigure 4). We chose the most proximal isoform, mod(mdg4)-64.2, which is encoded by the same strand as thecommon exons, and deduced a forward primer (ex4-virF),which exclusively hybridizes to common exon 4 of D. virilismod(mdg4) and a backward primer (64.2-B-RT), hybridiz-ing to the specific exon mod(mdg4)-64.2 of D. melanogaster(cf. Figure 2). The latter primer, despite three nucleo-tide substitutions, hybridizes to the orthologous D. virilisexon at the annealing temperature used (cf. materialsand methods). The two orthologous mod(mdg4)-64.2specific exons contain several nucleotide substitutionswithin the amplified region, which includes the positionof a single PvuII restriction site (Figure 4A). Restrictionof the resulting RT-PCR fragments with PvuII producesan internal 474-bp fragment in the case of the chimericD. virilis/D. melanogaster mod(mdg4)-64.2 cDNA (Figure4B, lane 1) and a 660-bp internal fragment in the caseof D. virilis mod(mdg4)-64.2 cDNA (Figure 4B, lane 2).These two restriction fragments have been used as indi-cators for the two cDNAs. With an equimolar mixtureof both orthologous D. melanogaster mod(mdg4)-64.2 andD. virilis mod(mdg4)-64.2 cDNA clones used, only as tem-plate, the D. virilis fragment was detectable after restric-tion with PvuII (Figure 4B, lane 3). The same result wasobtained when mixed RNA isolated from D. melanogasterand D. virilis was used as RT-PCR template, indicatingthe absence of significant template switching.

Chimeric transcripts could be detected when RNA

TA

BL

E1

Com

plem

enta

tion

anal

ysis

ofm

od(m

dg4)

alle

les

inth

epr

esen

ceof

D.

vir

mod

(mdg

4)tr

ansg

enes

Ch

rom

osom

allo

cati

onof

the

tran

sgen

e

23

32

P(w

�D

vm

od(m

dg4)

11.5

kb)/

�;

P(w

�D

vm

od(m

dg4)

11.5

kb)

P(w

�D

vm

od(m

dg4)

11.5

kb)

P(w

�D

vm

od(m

dg4)

6.8k

b)/�

;G

enot

ype/

fem

ales

,m

ales

mod

(mdg

4)02

/TM

3,Sb

Ser

mod

(mdg

4)02

/TM

3,Sb

Ser

mod

(mdg

4)ne

o129

/TM

3,Sb

Ser

mod

(mdg

4)02

/TM

3,Sb

Ser

mod

(mdg

4)02

/TM

3,Sb

Ser

sl(2

6.6%

/673

)sv

(79,

0%/8

50)

�(1

03.9

%/8

58)

sl(1

0.7%

/461

)m

od(m

dg4)

neo1

29/T

M3,

SbSe

rsv

(95.

1%/6

87)

�(1

03.6

%/8

75)

sv(9

2%/7

29)

sl(3

4%/4

13)

mod

(mdg

4)R

32/T

M3,

SbSe

rasl

f /sv

m(2

6.2%

/91.

8%/7

65)

slf /

svm

(37.

8%/9

5.9%

/693

)sl

f /sv

m(4

0.5%

/63.

2%/1

137)

�f /

svm

(4.0

%/5

2.3%

/762

)m

od(m

dg4)

07/T

M3,

SbSe

rsl

(12.

5%/6

35)

sl(5

.4%

/379

)�

(0.6

%/5

39)

�(0

%/4

36)

P(w

�D

vm

od(m

dg4)

11.5

kb)/

�;

mod

(mdg

4)02

/TM

3,Sb

Ser

�(1

06.7

%/1

097)

ND

ND

sv(7

4.0%

/613

)

Th

eP(

w�

Dv

mod

(mdg

4)11

.5kb

Not

I)tr

ansg

ene

enco

des

com

mon

exon

s1–

4an

dth

em

ostp

roxi

mal

spec

ific

exon

sm

od(m

dg4)

-642

,mod

(mdg

4)-6

01,m

od(m

dg4)

-535

,mod

(mdg

4)-

551,

and

mod

(mdg

4)-5

31,w

her

eas

the

tran

sgen

eP(

w�

Dv

mod

(mdg

4)6.

8kb

Not

I-X

baI)

enco

des

excl

usiv

ely

the

com

mon

exon

s1–

4.Fo

rvi

abili

ty:�

com

plem

enta

tion

;sv,

subv

ital

(i.e

.,on

aver

age

10

0an

d�

50%

ofth

eex

pect

edpr

ogen

ysu

rviv

eto

eclo

seas

adul

ts);

sl,

sem

ileth

al(i

.e.,

onav

erag

e5–

50%

ofth

eex

pect

edpr

ogen

ysu

rviv

eto

eclo

seas

adul

ts);

�,n

oco

mpl

emen

tati

on(

5%of

the

expe

cted

prog

eny

surv

ive

toec

lose

asad

ults

);th

epe

rcen

tage

ofex

pect

edh

omo-

,tra

ns-h

eter

ozyg

ous

offs

prin

g/to

tal

num

ber

offl

ies

issh

own

inpa

ren

thes

es.

aV

iabi

lity

oftr

ans-h

eter

ozyg

ous

offs

prin

gfe

mal

es(f

)an

dm

ales

(m)

issh

own

.

from females containing one copy of the second chro-


mosomal or the third chromosomal P(w� Dv mod(mdg4)11.5kb NotI) transgene was used as template for RT-PCR(Figure 4B, lanes 5 and 6, respectively). Also in 3-P(w�

Dv mod(mdg4) 11.5kb NotI) mod(mdg4)neo129 homozygotesand in 2-P(w� Dv mod(mdg4) 11.5kb NotI)/�; mod(mdg4)02/mod(mdg4)02, the chimeric transcript mod(mdg4)-64.2 isclearly detectable (Figure 4B, lane 7 and 8, respectively).The increased ratio of the chimeric 474-bp fragment infemales containing two copies of the transgene (Figure4B, lane 7) indicates dosage-dependent accumulationof the chimeric transcript. The smaller chimeric-specificfragment of 311 bp is not visible in most lanes becauseof the underrepresentation of the corresponding ampli-con. However, when an equimolar mixture of D. virilisand chimeric D. vir./D. mel. mod(mdg4)-64.2 cDNA clonesis used as template, all expected fragments, includingthe 311-bp fragment, are clearly visible (Figure 4B,lane 9).

Next we proved if isoforms not encoded by the D. virilistransgene are produced as chimeric transcripts. We usedthe D. virilis specific forward primer ex4-vir-F and threeD. melanogaster specific backward primers, mod(mdg4)-51.4-RT-B, mod(mdg4)-56.3-RT-B, and mod(mdg4)-67.2-RT-B. In three independent RT-PCR experiments withRNA from females of the genotype 2-P(w� Dv mod(mdg4)11.5kb NotI)/�; mod(mdg4)02/mod(mdg4)02, fragments ofthe expected size were obtained and sequencing revealedthe expected chimeric cDNAs. This result suggests thatobviously all Mod(mdg4) isoforms can be produced by in-terspecies trans -splicing. We conclude that important fea-tures involved in trans-splicing of mod(mdg4) are evolution-ary conserved between D. melanogaster and D. virilis.

The semiquantitative RT-PCR experiments describedabove do not allow an accurate estimation of the fre-Figure 4.—RT-PCR analysis demonstrating the expression

of D. virilis mod(mdg4)-64.2 and the chimeric D. virilis/D. mela- quency of interspecies mRNA trans-splicing. Thereforenogaster mod(mdg4)-64.2 transcript in transgenic D. melanogaster we cloned the resulting mod(mdg4)-64.2 RT-PCR frag-females. (A) Schematic of the RT-PCR assay to determine the ments obtained from homozygous 3-P(w� Dv mod(mdg4)ratio of both transcripts. (Top) Relevant mod(mdg4) transcripts

11.5kb NotI) females in three independent experiments(wavy bars; TSS represents the trans-splice site) encoded fromin pGEM-T and tested altogether 282 individual clonesthe endogenous D. melanogaster (Dm) locus (red bar) and

from the D. virilis (Dv) transgene (black bar) are shown. Right- for their identity via specific primer pairs (cf. materialsangled arrows indicate independent initiation of transcrip- and methods). Three of these clones were proved totion. cDNA fragments obtained after RT-PCR (solid bars) and be chimeric, whereas the remaining 279 represent D.restriction fragments resulting from digestion with the endo-

virilis cDNA clones. All chimeric clones and 59 of thenuclease PvuII (thin lines) are shown below the boldfaceD. virilis clones have been confirmed by sequencing.arrow. For RT-PCR, a forward primer specific for D. virilis

exon 4 and a backward primer annealing to exon 5 of Also in these experiments no sign of template switchingmod(mdg4)-64.2 of both species were used. (B) Results of the was found. According to these results, the chimeric tran-RT-PCR to demonstrate the expression of the D. virilis and scripts compose �1% of the D. virilis mod(mdg4)-64.2the chimeric D. virilis/D. melanogaster mod(mdg4)-64.2 isoforms.

transcript encoded by the transgene. To confirm theseTemplates used for PCR are lane 1, chimeric cDNA clone D.results, we next performed real-time RT-PCR experi-vir./D. mel. mod(mdg4)-64.2 ; lane 2, cDNA clone D. vir. mod

(mdg4)-64.2 ; lane 3, equimolar mixture of D. mel. mod(mdg4)- ments.64.2 and D. vir. mod(mdg4)-64.2 cDNA clones; lane 4, 100-bp First we determined the expression level of D. melano-ladder; lanes 5 and 6, RT templates of transgenic females with gaster mod(mdg4) with respect to ribosomal protein 49the genotypes 2-P(w� Dv mod(mdg4) 11.5kb Not I)/� and 3-P(w�

mRNA (rp49) with a specific primer pair deduced fromDv mod(mdg4) 11.5kb Not I)/�; lanes 7 and 8, 2-P(w� Dv modmod(mdg4) common exons using total RNA isolated(mdg4) 11.5kb Not I) mod(mdg4)neo129 homozygotes and 3-P(w�

Dv mod(mdg4) 11.5kb Not I)/�; mod(mdg4)02/mod(mdg4)02; lane from w1118 females as control. According to these re-9, mixture of D. vir. mod(mdg4)-64.2 cDNA and chimeric D. vir./ sults, mod(mdg4) expression is 76-fold higher than thatD. mel. mod(mdg4)-64.2 cDNA in a ratio of 1:1. of rp49 (Table 2). The two specific D. melanogaster iso-


TABLE 2

Results of the real-time quantitative RT-PCR

GenotypeRelative expression(normalized to rp49a) w 1118 (control) w 1118 3-P(w� Dv 11.5kb) w 1118 2-P(w� Dv 6.8kb)

Common exons 1–4D. mel. 76.0 12.1 75.6 13.8 44.2 5.4D. vir. — 59.2 12.7 27.8 6.2

Specific isoformsD. mel. 64.2 0.667 0.042 0.314 0.044 0.132 0.010D. vir. 64.2 (transgenic) — 1.200 0.178 —D. vir./D. mel. 64.2 (chimeric) — 0.008 0.001 0.005 0.001D. mel. 67.2 0.654 0.085 0.451 0.051 0.214 0.013D. vir./D. mel. 67.2 (chimeric) — 0.021 0.004 0.025 0.003

For this experiment RNA was extracted from adult females of w 1118, w 1118; 3-P(w� Dv mod(mdg4) 11.5kb Not I)/3-P(w� Dv mod(mdg4) 11.5kb Not I) and w 1118; 2-P(w� Dv mod(mdg4) 6.8kb Not I-XbaI)/2-P(w� Dv mod(mdg4) 6.8kbNot I-XbaI).

a rp49 � 1.

forms, mod(mdg4)-64.2 and mod(mdg4)-67.2, are rep- cific primer pairs have been deduced. Real-time RT-PCR experiments indicate that chimeric D. virilis/D. mel-resented with 1% each with respect to mod(mdg4)

common exons. It has to be noted that expression of anogaster mod(mdg4)-64.2 represents 2.5% with respect tothe corresponding endogenous D. melanogaster isoformthese isoforms is still in the range of rp49 expression.

At least mod(mdg4)-67.2 is supposed to be one of the (0.008/0.314, Table 2), whereas the chimeric mod(mdg4)-67.2 isoform represents 4.7% of the corresponding en-most abundant mod(mdg4) isoforms (Gerasimova et al.

1995; Buchner et al. 2000) and therefore to be expected dogenous transcript (0.021/0.451, Table 2). These re-sults indicate that chimeric transcripts are produced atat significantly higher expression levels compared to

most other mod(mdg4) isoforms. However, if trans-splic- a significant level. The frequency of chimeric mod(mdg4)-64.2 is in a similar range as determined by analyzinging is the main mechanism for generating all mod(mdg4)

isoforms (Dorn and Krauss 2003), the transcript con- individual RT-PCR clones.Next we performed the same RT-PCR experimentstaining common exons 1–4, which is used as splice do-

nor for all isoforms, should be expressed at a high level. using RNA isolated from w1118; P(w� Dv mod(mdg4) 6.8kbNotI-XbaI)/P(w� Dv mod(mdg4) 6.8kb NotI-XbaI) females.Next we used RNA isolated from w1118 females con-

taining two copies of the third chromosomal P(w� Dv Expression of D. virilis and the endogenous commonexons as well as endogenous isoforms mod(mdg4)-64.2mod(mdg4) 11.5kb NotI) transgene as template for real-

time qRT-PCR. The endogenous D. melanogaster com- and -67.2 is decreased compared to P(w� Dv mod(mdg4)11.5kb NotI) transgenic females. The ratio of the twomon exons are expressed at almost the same level (75.6,

Table 2) as in the control females, whereas the trans- corresponding chimeric transcripts is increased approx-imately twofold (3.8 and 11.7%, respectively). This in-genic D. virilis common exons (59.2) are expressed at a

slightly lower level (Table 2). In these transgenic females crease could be explained by the absence of the D.virilis specific exons in the short transgene trappingthe isoform mod(mdg4)-64.2 is expressed from the en-

dogenous mod(mdg4) locus and from the D. virilis trans- a significant fraction of the common exons as splicedonor.gene. Whereas expression of the D. melanogaster isoform

is decreased by a factor of 2 compared to control fe- We conclude from these experiments that proteinsproduced from chimeric mod(mdg4) transcripts in trans-males (0.314 vs. 0.667, respectively), the transgenic D.

virilis orthologous isoform is increased by a factor of 2 genic P(w� Dv mod(mdg4) 6.8kb NotI-XbaI) flies are suffi-cient to rescue viability at least partially. The improved(1.2). Isoform mod(mdg4)-67.2 is not encoded by the D.

virilis transgene. Also in this case expression is decreased rescue ability of the P(w� Dv mod(mdg4) 11.5kb NotI)transgene can thus be explained by the additional ex-in females with two copies of the P(w� Dv mod(mdg4)

11.5kb NotI) transgene (0.451 vs. 0.654). It is not clear pression of the five proximal orthologous D. virilisMod(mdg4) isoforms encoded by this transgene.whether these differences reflect changes in general

transcriptional activity in both genotypes or whether Mod(mdg4) staining pattern of polytene chromo-somes of mutant larvae is restored in the presence offeedback mechanisms regulating the mod(mdg4) expres-

sion are involved. the D. virilis transgene: Previously, we demonstrated thatMod(mdg4) proteins are not detected on salivary glandTo determine the ratio of chimeric transcripts, spe-


Figure 5.—The D. virilis mod(mdg4) trans-gene partially rescues the Mod(mdg4)staining pattern on polytene chromosomesof homozygous mod(mdg4) mutant larvae.Staining of polytene chromosomes of thirdinstar larvae with genotypes 2-P(w� Dv mod(mdg4) 11.5kb NotI); mod(mdg4)02/mod(mdg4)02

(A–C), w1118 (D–F), 2-P(w� Dv mod(mdg4)11.5kb NotI); mod(mdg4)02/mod(mdg4)02 (G,H, and J), and mod(mdg4)02/mod(mdg4)02 lar-vae not containing the transgene (K–M).(A, D, G, and K) Propidiumijodid stain-ing. (B) Staining with anti-Mod(mdg4)-58.0BTB-534. (E, H, and L) Staining with anti-Mod(mdg4)-67.2403-610. (C, F, J, and M)Merged images. (N) Comparison of thestaining pattern of the tip of the X chromo-some of w 1118 and 2-P(w� Dv mod(mdg4)11.5kb Not I); mod(mdg4)02/ mod(mdg4)02

larvae.

polytene chromosomes of homozygous mod(mdg4)02 (Figure 5, B). Similar results were obtained for other trans-heterozygotes. Next we used the specific antibody anti-third larval stages (Buchner et al. 2000). To prove if,

in the presence of the D. virilis transgene, binding of Mod(mdg4)-67.2403-610, detecting exclusively this iso-form. The staining pattern of polytene chromosomesMod(mdg4) proteins is restored, we performed immu-

nostaining of 2-P(w� Dv mod(mdg4) 11.5kb NotI)/�; of 2-P(w� Dv mod(mdg4) 11.5kb NotI); mod(mdg4)02/mod(mdg4)02 larvae is similar to that of mod(mdg4)� chro-mod(mdg4)02/mod(mdg4)02 larvae with anti-Mod(mdg4)-

58.0BTB-534, an antiserum detecting all Mod(mdg4) iso- mosomes (Figure 5, E and H), indicating that significantlevels of chimeric Mod(mdg4)-67.2 protein are pro-forms. Independent experiments clearly indicate signifi-

cant immunostaining in the presence of the transgene duced. In mod(mdg4)02 homozygous larvae, no staining


unusual type of gene structure in D. melanogasterprompted us to analyze the orthologous locus from thedistantly related species D. virilis. It represents an evolu-tionarily distant species that was separated �40–60 mil-lion years ago from the Sophophora, which includes D.melanogaster. This period of time allowed for the selec-tion of functionally essential genes. A number of or-thologous genes have been studied in detail and theirfunctional conservation in D. virilis was demonstratedby mutant rescue experiments (Kassis et al. 1986; Colotet al. 1988; Hart et al. 1993; Bopp et al. 1996). Thedegree of the overall conservation within coding regionsFigure 6.—Western blot analysis of dissected salivary glandsis variable and can reach up to 98% similarity (Tomi-with anti-Mod(mdg4)-67.2403-610 antibody. Lane 1, w 1118; lane 2,

mod(mdg4)02/mod(mdg4)02; lane 3, 2-P(w� Dv mod(mdg4) 11.5kb naga et al. 1992). Our results demonstrate a strongNot I); mod(mdg4)02/mod(mdg4)02; lane 4, 3-P(w� Dv mod(mdg4) evolutionary conservation of all Mod(mdg4) isoforms6.8kb Not I-XbaI)/�; mod(mdg4)02/mod(mdg4)02. Antitubulin anti- identified in D. virilis, indicating the functional signifi-body staining was used as loading control.

cance of the multiple isoforms. We previously presentedevidence for a functional differentiation of at least twoisoforms, Mod(mdg4)-58.0 and Mod(mdg4)-67.2 in D.is detected (Figure 5L). To prove if all Mod(mdg4)-

67.2 binding sites detected in wild-type larvae are also melanogaster (Buchner et al. 2000). The high degree ofsequence conservation of both isoforms in D. virilis isfound in transgenic 2-P(w� Dv mod(mdg4) 11.5kb NotI);

mod(mdg4)02/mod(mdg4)02 larvae, we analyzed a selected in good agreement with binding to corresponding siteson polytene chromosomes as shown for isoform Modregion on the X chromosome by comparing the appro-

priate staining pattern (Figure 5N). At least in this re- (mdg4)-58.0. Its binding to corresponding subdivisionson polytene chromosomes suggests an involvement ingion all strong binding sites correspond to each other,

indicating both the reestablishment of the staining pat- regulation of a subset of orthologous genes in D. melano-gaster and D. virilis.tern of Mod(mdg4)-67.2 and the binding specificity of

the chimeric protein in transgenic larvae. However, use The common N-terminal region, which is part of allisoforms and therefore supposed to contribute generalof the antibody detecting all isoforms, the staining pat-

tern, and the signal intensity were more variable. We functions, shows an extended identity beyond the BTB/POZ domain. This common protein region representswere not able to perform staining of polytene chromo-

somes of 2-P(w� Dv mod(mdg4) 6.8kb NotI-XbaI)/�; about two-thirds of any of the Mod(mdg4) proteins.Gause et al. (2001) have shown that the ubiquitouslymod(mdg4)02/mod(mdg4)02 larvae because salivary glands

and nuclei were reduced in size and changed chromo- expressed protein Chip interacts with the common re-gion of Mod(mdg4) in D. melanogaster. Chip is supposedsome morphology prevented reproducible antibody

staining. To determine if the specific antibody detects to facilitate enhancer-promoter interactions in a largenumber of genes and was shown to interact geneticallythe expected full-length chimeric Mod(mdg4)-67.2 pro-

tein, we performed Western analysis with salivary glands and physically with several LIM- and homeodomain-containing transcription factors (Morcillo et al. 1997;of third instar 2-P(w� Dv mod(mdg4) 11.5kb NotI);

mod(mdg4)02/mod(mdg4)02 and 2-P(w� Dv mod(mdg4) 6.8kb Torigoi et al. 2000; Heitzler et al. 2003). These data,together with the observed pleiotropic mutant effectsNotI-XbaI)/�; mod(mdg4)02/mod(mdg4)02 larvae (Figure 6).

A protein with the same molecular weight as in isogenic of most mod(mdg4) mutants, indicate a putative link be-tween the several hundred binding sites of Mod(mdg4)w1118 larvae as the control for mod(mdg4)� (Figure 6,

lane 1) could be clearly detected in both transgenic on polytene chromosomes and their involvement intranscriptional regulation of a large number of genes.mod(mdg4)02/mod(mdg4)02 genotypes (Figure 6, lanes 3 and

4) whereas in mod(mdg4)02 homozygotes the protein is The strong conservation of the common protein regionin both Drosophila species might be the consequenceabsent (Figure 6, lane 2). The protein level is decreased

in transgenic mutant larvae. However, our results indi- of the evolutionarily conserved interaction with Chipand other putative interacting proteins. The N-terminalcate that proteins encoded by chimeric transcripts that

are generated from pre-mRNAs transcribed from non- BTB/POZ domain is almost identical in both species.This domain was shown to mediate homo- and/or hetero-homologous chromosomes can be produced in a sig-

nificant amount. dimerization (Bardwell and Treisman 1994). A simi-lar degree of conservation between D. melanogaster andD. virilis was found for the BTB/POZ domain containing

DISCUSSIONgene GAGA/Trl (Lintermann et al. 1998). Also in thiscase at least two alternatively spliced isoforms containingThe limited knowledge of the functional significance

of the large number of mod(mdg4) isoforms and the a common N-terminal region of 400 amino acids but


variable C termini have been described. However, in of chimeric transcripts in vivo. The identification ofchimeric mod(mdg4) isoforms in transgenic flies clearlycontrast to mod(mdg4), no significant functional differ-

entiation between the two GAGA isoforms has been indicates that the mechanism of mRNA trans-splicingis conserved between the distantly related Drosophiladescribed (Soeller et al. 1993; Benyajati et al. 1997).

If specific C termini of orthologous Mod(mdg4) iso- species. Quantitative RT-PCR experiments reveal thatin case of isoform Mod(mdg4)-67.2 the chimeric D.forms are compared, a remarkable degree of identity

within the FLYWCH domain, a Cys2His2-motif-contain- virilis/D. melanogaster transcript in transgenic flies con-taining two copies of the second chromosomal P(w� Dving protein domain, is found. This domain is supposed

to be involved in protein-protein interactions (Dorn mod(mdg4) 6.8kb NotI-XbaI) transgene represents �12%of the corresponding endogenous D. melanogaster tran-and Krauss 2003 and references therein). Strong con-

servation of most amino acid positions within this motif script. The Mod(mdg4)-67.2 protein can be clearly de-tected on polytene chromosomes of 2-P(w� Dv modbetween orthologous isoforms implies their functional

importance for isoform-specific interactions with other (mdg4) 11.5kb NotI)/�; mod(mdg4)02/mod(mdg4)02 larvaebut not in mod(mdg4)02 homozygous larvae. Because theproteins. The unique C-terminal region of isoform

Mod(mdg4)-67.2 has been demonstrated to interact specific mod(mdg4)-67.2 exons are not encoded by theD. virilis transgene, this result strongly suggests thatwith Su(Hw) to create a functional gypsy insulator ele-

ment (Gause et al. 2001; Ghosh et al. 2001) whereas the cytologically detected protein represents the chime-ric D. virilis/D. melanogaster Mod(mdg4)-67.2 protein,the unique C terminus of isoform Mod(mdg4)-56.3/

Doom interacts with the baculovirus inhibitor of apopto- which is produced in a significant amount. In fact, thepresence of considerable amounts of the full-lengthsis protein/IAP (Harvey et al. 1997). The high degree

of sequence identity suggests that these interactions are Mod(mdg4)-67.2 protein was demonstrated in Westernblot analysis. The maintenance of the binding patternconserved in D. virilis. If the orthologous D. virilis iso-

forms Mod(mdg4)-64.2, Mod(mdg4)-60.1, and Mod of the chimeric Mod(mdg4)-67.2 isoform compared tothe D. melanogaster Mod(mdg4)-67.2 on polytene chro-(mdg4)-67.2 are compared with their counterparts in

D. melanogaster, it becomes evident that additional amino mosomes also implicates the functional conservation ofthe D. virilis N-terminal region.acid positions flanking the FLYWCH motif are highly

conserved. However, the extension and the location Recently, Mongelard et al. (2002) demonstrated thatinterallelic complementation is facilitated by mRNAof the identity beyond the FLYWCH motif is isoform

dependent. In case of Mod(mdg4)-67.2, an additional trans-splicing if two mutations disrupting independentmod(mdg4) mRNAs are combined in trans. They assumestrongly conserved sequence motif of 22 amino acids is

located at the C terminus. On the basis of pull-down that the close proximity of donor and acceptor mRNAswithin the mod(mdg4) locus is a prerequisite for genera-experiments with a C-terminal truncated (deletion of

43 amino acids) Mod(mdg4)-67.2 protein and the ob- tion of significant amounts of wild-type Mod(mdg4)-67.2 protein. The lola locus of D. melanogaster representsserved phenotype connected with the corresponding

mutant protein (Mod(mdg4)-67.2T6) the FLYWCH do- a second complex gene in which mRNA trans-splicingwas demonstrated (Horiuchi et al. 2003). Mutationsmain itself is not sufficient for interaction with Su(Hw)

(Gause et al. 2001), indicating the functional impor- interfering with the pairing of the lola locus reducethe in vivo trans-splicing of isoform T from 44 to 1%.tance of the strongly conserved 22 C-terminal amino

acids. Also, the isoforms without the FLYWCH motif are However, the authors did not prove the consequenceson a protein level. Our transgene assay clearly demon-conserved as shown for Mod(mdg4)-58.0 (identity of

51% within the unique C terminus). Recently, an evolu- strates that even underrepresented chimeric transcriptsproduced from mRNAs encoded by nonhomologoustionary analysis of several Dipteran orthologous mod

(mdg4) loci revealed a significant conservation of most chromosomes can produce considerable levels of thecorresponding protein. Mutant rescue experiments withisoforms, including Mod(mdg4)-58.0, Mod(mdg4)-60.1,

Mod(mdg4)-64.2, and Mod(mdg4)-67.2 (Labrador two different D. virilis mod(mdg4) transgenes indicate thefunctional conservation of Mod(mdg4) protein iso-and Corces 2003; Krauss and Dorn 2004).

Two conclusions can be drawn from the evolutionary forms. Both the P(w� Dv mod(mdg4) 11.5kb NotI) trans-gene, which encodes the five proximal isoforms, and theconservation of Mod(mdg4) proteins. First, the large

number of isoforms is functionally important in both P(w� Dv mod(mdg4) 6.8kb NotI-XbaI) transgene, encodingexclusively common exons 1–4, facilitate rescue of reces-Drosophila species and second, the conservation of the

unique C-terminal regions clearly points to a functional sive lethality of mod(mdg4) mutant alleles. We supposethat the rescue ability of the short transgene dependsdifferentiation between single isoforms.

In the present study we demonstrate for the first time mainly on its capacity to produce sufficient chimerictranscripts consisting of the D. virilis common exonsthat along with the evolutionary conservation of the

unusual gene structure of mod(mdg4) in D. virilis mRNA and the endogenous D. melanogaster specific exons,which was demonstrated at least for isoform Modtrans-splicing is also conserved in both species. We per-

formed three different assays to prove the existence (mdg4)-67.2. However, the significantly reduced rescue


Dorn, R., V. Krauss, G. Reuter and H. Saumweber, 1993 Theability of the shorter transgene indicates that all or someenhancer of position-effect variegation of Drosophila, E(var)3–

isoforms have to exceed a critical threshold to restore 93D, codes for a chromatin protein containing a conserved do-main common to several transcriptional regulators. Proc. Natl.viability completely. The P(w� Dv mod(mdg4) 11.5kb NotI)Acad. Sci. USA 90: 11376–11380.transgene, which produces five orthologous D. virilis

Dorn, R., G. Reuter and A. Loewendorf, 2001 Transgene analysisisoforms, significantly improves rescue ability. We can- proves mRNA trans-splicing at the complex mod(mdg4) locus in

Drosophila. Proc. Natl. Acad. Sci. USA 98: 9724–9729.not exclude position effects influencing the expressionGause, M, P. Morcillo and D. Dorsett, 2001 Insulation of en-level of the transgene. Further experiments with a series

hancer-promoter communication by a gypsy transposon insert inof independent insertions of the short transgene scat- the Drosophila cut gene: cooperation between suppressor of

hairy-wing and modifier of mdg4 proteins. Mol. Cell. Biol. 21:tered throuhgout the genome should provide further4807–4817.insight into a putative correlation of genomic transgene

Gerasimova, T. I., D. A. Gdula, D. V. Gerasimov, O. Simonova andposition and efficiency of trans-splicing. V. G. Corces, 1995 A Drosophila protein that imparts direction-

ality on a chromatin insulator is an enhancer of position-effectThe observed frequency of chimeric transcripts, al-variegation. Cell 82: 587–597.though significantly lower as compared to the corre-

Ghosh, D., T. I. Gerasimova and V. G. Corces, 2001 Interactionssponding endogenous transcript, can be interpreted in between the Su(Hw) and Mod(mdg4) proteins required for gypsy

insulator function. EMBO J. 20: 2518–2527.two ways. First, the splice donor containing the D. virilisGorczyca, M., E. Popova, X. X. Jia and V. Budnik, 1999 The genemod(mdg4) common exons is produced at a high level,

mod(mdg4) affects synapse specificity and structure in Drosophila.enabling its spreading in the nucleus. Thus a significant J. Neurobiol. 39: 447–460.

Graveley, B. R., 2001 Alternative splicing: increasing diversity innumber of donor molecules are in close proximity tothe proteomic world. Trends Genet. 17: 100–107.mod(mdg4) acceptor mRNAs, even if they are transcribed

Hart, A. C., S. D. Harrison, D. L. Van Vactor, G. M. Rubin andfrom a nonhomologous chromosome. The much higher S. L. Zipursky, 1993 The interaction of bride of sevenless with

sevenless is conserved between Drosophila virilis and Drosophila mela-expression of the common exons compared to the spe-nogaster. Proc. Natl. Acad. Sci. USA 9: 5047–5051.cific isoform mod(mdg4)-67.2 in w1118 females (116-fold,

Harvey, A. J., A. P. Bidwai and L. K. Miller, 1997 Doom, a productcf. Table 2) is in agreement with this hypothesis. A sec- of the Drosophila mod(mdg4) gene, induces apoptosis and binds

to baculovirus inhibitor-of-apoptosis proteins. Mol. Cell. Biol. 17:ond explanation supposes transcription of both precur-2835–2843.sor mRNAs within the same compartment of the nucleus

Heitzler, P., L. Vanolst, I. Biryukova and P. Ramain, 2003 En-(for review, see Cockell and Gasser 1999), thereby hancer-promoter communication mediated by Chip during Pan-

nier-driven proneural patterning is regulated by Osa. Genes Dev.increasing the frequency of chimeric mRNAs.17: 591–596.

We thank P. Geyer, T. Parnell, and B. Gilmore for the generous Horiuchi, T., E. Ginigert and T. Aigaki, 2003 Alternative trans-gift of the specific antibody anti-Mod(mdg4)-67.2403-610, Anja Ebert for splicing of constant and variable exons of a Drosophila axonhelp in immunocytology, M. Kube for technical assistance, and K. guidance gene, lola. Genes Dev. 17: 2496–2501.

Jones, C. W., and M. W. Dalton, 1991 Interspecific comparison ofKittlaus for sequence analyis. We are indebted to S. Phalke for criticalthe structure and regulation of the Drosophila ecdysone-induc-reading of the manuscript. This work was supported by a grant fromible gene E74. Genetics 127: 535–543.the Deutsche Forschungsgemeinschaft to R.D. (Do407/2-2).

Kassis, J. A., S. J. Poole, D. K. Wright and P. H. O’Farrell, 1986Sequence conservation in the protein coding and intron regionsof the engrailed transcription unit. EMBO J. 5: 3583–3589.

Klementz, R., U. Weber and W. J. Gehring, 1986 The white geneLITERATURE CITEDas a marker in a new P-element vector for gene transfer in Dro-sophila. Nucleic Acids Res. 10: 3947–3959.Bardwell, V. J., and R. Treisman, 1994 The POZ domain: a con-

served protein-protein interaction motif. Genes Dev. 8: 1664– Krauss, V., and R. Dorn, 2004 Evolution of the trans-splicing Dro-sophila locus mod(mdg4) in several species of Diptera and Lepidop-1677.

Benyajati, C., L. Mueller, N. Xu, M. Pappano, J. Gao et al., tera. Gene 331: 165–176.Kress, H., 2001 Evolution of 5S rRNA gene families in Drosophila.1997 Multiple isoforms of GAGA factor, a critical component

of chromatin structure. Nucleic Acids Res. 25: 3345–3353. Chromsome Res. 9: 403–415.Labrador, M., and V. G. Corces, 2003 Extensive exon reshufflingBlumenthal, T., 1998 Gene clusters and polycistronic transcription

in eukaryotes. BioEssays 20: 480–487. over evolutionary time coupled to trans-splicing in Drosophila.Genome Res. 13: 2220–2228.Bopp, D., G. Calhoun, J. I. Horabin, M. Samuels and P. Schedl,

1996 Sex-specific control of Sex-lethal is a conserved mecha- Labrador, M., F. Mongelard, P. Plata-Rengifo, E. M. Baxter,V. G. Corces et al., 2001 Protein encoding by both DNA strands.nism for sex determination in the genus Drosophila. Develop-

ment 122: 971–982. Nature 409: 1000.Lanio, W., U. Swida and H. Kress, 1994 Molecular cloning of theBuchner, K., P. Roth, G. Schotta, V. Krauss, H. Saumweber et al.,

2000 Genetic and molecular complexity of the position effect Drosophila virilis larval glue protein gene Lgp-3 and its comparativeanalysis with other Drosophila glue protein genes. Biochim. Bio-variegation modifier mod(mdg4) in Drosophila. Genetics 155: 141–

157. phys. Acta 1219: 576–580.Lindsley, D. L., and G. G. Zimm, 1992 The Genome of DrosophilaCockell, M., and S. M. Gasser, 1999 Nuclear compartments and

gene regulation. Curr. Opin. Genet. Dev. 9: 199–205. melanogaster. Academic Press, New York.Lintermann, K.-L., G. E. Roth, K. King-Jones, G. Korge and M.Colot, H. V., J. C. Hall and M. Rosbash, 1988 Interspecific com-

parison of the period gene of Drosophila reveals large blocks of Lehmann, 1998 Comparison of the GAGA factor genes of Dro-sophila melanogaster and Drosophila virilis reveals high conservationnonconserved coding DNA. EMBO J. 7: 3929–3937.

DiBello, P. R., D. A. Withers, C. A. Bayer, J. W. Fristrom and of GAGA factor structure beyond the BTB/POZ and DNA-bind-ing domains. Dev. Genes Evol. 208: 447–456.G. M. Guild, 1991 The Drosophila Broad-Complex encodes a

family of related proteins containing zinc fingers. Genetics 129: Madden, K., D. Crowner and E. Giniger, 1999 LOLA has theproperties of a master regulator of axon-target interaction for385–397.

Dorn, R., and V. Krauss, 2003 The modifier of mdg4 locus in Drosoph- SNb motor axons of Drosophila. Dev. Biol. 213: 301–313.Maniatis, T., and B. Tasic, 2002 Alternative pre-mRNA splicingila: functional complexity is resolved by trans-splicing. Genetica

117: 165–177. and proteome expansion in metazoans. Nature 418: 236–243.


Mongelard, F., M. Labrador, E. M. Baxter, T. I. Gerasimova and Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon-ing: A Laboratory Manual. Cold Spring Harbor Laboratory Press,V. G. Corces, 2002 Trans-splicing as a novel mechanism toCold Spring Harbor, NY.explain interallelic complementation in Drosophila. Genetics

Soeller, W. C., C. E. Oh and T. B. Kornberg, 1993 Isolation of160: 1481–1487.cDNAs encoding the Drosophila GAGA transcription factor. Mol.Morcillo, P., C. Rosen, M. K. Baylies and D. Dorsett, 1997 Chip,Cell. Biol. 13: 7961–7970.a widely expressed chromosomal protein required for segmenta-

Tamate, H. B., R. C. Patel, A. E. Riedl and M. Jacobs-Lorena, 1990tion and activity of a remote wing margin enhancer in Drosophila.Overproduction and translational regulation of rp49 ribosomalGenes Dev. 11: 2729–2740.protein mRNA in transgenic Drosophila carrying extra copies ofPimpinelli, S., S. Bonaccorsi, L. Fanti and M. Gatti, 2000 Drosoph-the gene. Mol. Gen. Genet. 221: 171–175.ila: A Laboratory Manual, edited by W. Sullivan, M. Ashburner

Tominaga, H., T. Shiba and S. Narise, 1992 Structure of Drosophilaand S. Hawley. Cold Spring Harbor Laboratory Press, Plainview,virilis glycerol-3-phosphate dehydrogenase gene and a comparison withNY.the Drosophila melanogaster gene. Biochim. Biophys. Acta 1131:Read, D., and J. L. Manley, 1992 Alternatively spliced transcripts233–238.of the Drosophila tramtrack gene encode zinc finger proteins with

Torigoi, E., I. M. Bennani-Baiti, C. Rosen, K. Gonzalez, P. Mor-distinct DNA binding specificities. EMBO J. 11: 1035–1044.cillo et al., 2000 Chip interacts with diverse homeodomainRead, D., M. J. Butte, A. F. Dernburg, M. Frasch and T. B. Korn- proteins and potentiates bicoid activity in vivo. Proc. Natl. Acad.

berg, 2000 Functional studies of the BTB domain in the Dro- Sci. USA 97: 2686–2691.sophila GAGA and Mod(mdg4) proteins. Nucleic Acids Res. 28: Wimber, D. E., and D. M. Steffenson, 1970 Localization of 5S RNA3864–3870. genes in Drosophila chromosomes by RNA-DNA hybridization.

Rubin, G. M., and A. C. Spradling, 1982 Genetic transformation Science 170: 639–641.of Drosophila with transposable element vectors. Science 218:348–353. Communicating editor: T. C. Kaufman

trans-splicing of the mod(mdg4) complex locus is ...the modiﬁer of mdg4, mod(mdg4), locus in...

Documents