Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10203-10207, November 1991 Genetics Evolution of gene position: Chromosomal arrangement and sequence comparison of the Drosophila melanogaster and Drosophila virilis sina and Rh4 genes (eye development/rhodopsin/seven in absentia/retroposon/gene evolution) THOMAS P. NEUFELD, RICHARD W. CARTHEW, AND GERALD M. RUBIN Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Contributed by Gerald M. Rubin, August 16, 1991 ABSTRACT The seven in absentia (sina) gene of Drosoph- ila encodes a nuclear protein required for normal eye devel- opment. In Drosophila melanogaster, the sina gene is located within an intron of the Rh4 opsin gene. We examine here the nucleotide sequences and chromosomal arrangements of these genes in Drosophila viris. An interspecies comparison between D. melanogaster and D. virus reveals that the protein-coding sequences of the sina and Rh4 genes are highly conserved, but the relative chromosomal position and structural arrangement of these genes differ between the two species. In particular, the sina and Rh4 genes are widely separated in D. virUs, and there is no intron in the Rh4 gene. Our results suggest that the Rh4 gene was translocated to another chromosomal location by a retrotransposition event. The physical relationships of genes on chromosomes may reflect both functional significance and evolutionary history. For example, genes of related function are often grouped into clusters (1-3). In many cases, such arrangements may simply reflect the mechanisms of duplication by which these genes arose. However, the potential for clustered genes to be coordinately influenced by shared regulatory elements or chromatin structures suggests that such arrangements may be functionally significant. The Antennapedia and Bithorax gene complexes, which have been conserved from Droso- phila to mammals (4, 5), are illustrative of arrangements that are likely under functional constraint. An unusual example of such clustering is demonstrated by two genes involved in the development and function of the Drosophila melanogaster visual system. The seven in ab- sentia (sina) gene encodes a nuclear protein required for the proper specification of the R7 photoreceptor cell (ref. 6; for review, see refs. 7 and 8). In flies mutant for sina, precursor cells that would normally develop into R7 neurons instead differentiate as nonneuronal cone cells. The sina gene is located within a 9-kilobase (kb) intron of the Rh4 gene, which encodes a photosensitive opsin (9). Interestingly, Rh4 is expressed exclusively in R7 photoreceptor cells, those cells that are missing in sina mutants. Although the intimate association of these genes is intriguing in light of their related functions, the significance of this arrangement is unclear. For example, the expression patterns of the two genes are not identical; Rh4 expression begins in the last stages of eye development and is restricted to the R7 cell (9), whereas the sina gene is expressed earlier and in a greater range of cell types (6). Additionally, the sina and Rh4 proteins are struc- turally dissimilar and have rather disparate roles in eye development and function. If the structural arrangement of the sina and Rh4 genes is functionally relevant, one might expect it to be conserved in other species. To examine this possibility, we cloned and sequenced the sina and Rh4 homologs from Drosophila virilis* and examined the physical organization of these genes in this species. Our results indicate that although the coding sequences of sina and Rh4 are highly conserved between D. virilis and D. melanogaster, the chromosomal arrangements of these genes are not. MATERIALS AND METHODS A A EMBL3 D. virilis genomic library (from M. Scott, Stanford University) was screened at reduced stringency with a 1.9-kb BamHI-Xho I fragment of D. melanogaster sina 2CI cDNA (6). Filters were hybridized at 450C in 5x standard saline citrate (SSC) (lx SSC is 150 mM NaCl/15 mM sodium citrate)/100 mM NaH2PO4, pH 6.8/0.1% poly- vinylpyrrolidone-40/0. 1% bovine serum albumin/0. 1% Ficoll/0.1% SDS/sonicated salmon sperm DNA at 100 pg/ml and washed at 50°C in 0.2x SSC/0.1% SDS. Eight positive clones were obtained from 105 plaques screened. A 2.8-kb Xho I-HindIII fragment of phage DNA that hybridized to the 2CI cDNA probe was identified by DNA blot analysis. This fragment was sonicated and subcloned into M13mplO for sequencing by the dideoxynucleotide chain-termination method (10). Sequences were analyzed by using IntelliGe- netics software. A 2.2-kb EcoRI D. virilis genomic DNA fragment contain- ing the Rh4 gene was isolated (ref. 11; and M. Fortini, personal communication). The nucleotide sequence of this fragment was determined as described above. Additional genomic DNA from the D. virilis Rh4 region was isolated by screening the A EMBL3 library with this fragment. Salivary gland polytene chromosomes were isolated from D. virilis third instar larvae and hybridized, as described by Zuker et al. (12). Probes were generated from 2.8-kb Xho I-HindIII and 2.2-kb EcoRI genomic fragments containing the sina and Rh4 genes, respectively. The probes were labeled by nick-translation with biotinylated dUTP and de- tected with streptavidin-conjugated horseradish peroxidase, by using the Detek I-HRP kit (Enzo Diagnostics) and then stained with diaminobenzidine. Chromosomes were hybrid- ized with each probe individually or both probes simultane- ously. RESULTS AND DISCUSSION Cloning and Sequencing of D. virUs sina and Rh4. The D. virilis sina gene was isolated by screening a D. virilis genomic library with a D. melanogaster sina cDNA, as described. In Fig. 1 the nucleotide and predicted amino acid sequences of D. virilis sina are displayed and compared with the D. melanogaster sina gene (6). The two predicted proteins are *The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M77281 and M77282). 10203 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Transcript
Proc. Natl. Acad. Sci. USAVol. 88, pp. 10203-10207, November 1991Genetics
Evolution of gene position: Chromosomal arrangement andsequence comparison of the Drosophila melanogaster andDrosophila virilis sina and Rh4 genes
(eye development/rhodopsin/seven in absentia/retroposon/gene evolution)
THOMAS P. NEUFELD, RICHARD W. CARTHEW, AND GERALD M. RUBINHoward Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Contributed by Gerald M. Rubin, August 16, 1991
ABSTRACT The seven in absentia (sina) gene of Drosoph-ila encodes a nuclear protein required for normal eye devel-opment. In Drosophila melanogaster, the sina gene is locatedwithin an intron of the Rh4 opsin gene. We examine here thenucleotide sequences and chromosomal arrangements of thesegenes in Drosophila viris. An interspecies comparison betweenD. melanogaster and D. virus reveals that the protein-codingsequences of the sina and Rh4 genes are highly conserved, butthe relative chromosomal position and structural arrangementof these genes differ between the two species. In particular, thesina and Rh4 genes are widely separated in D. virUs, and thereis no intron in the Rh4 gene. Our results suggest that the Rh4gene was translocated to another chromosomal location by aretrotransposition event.
The physical relationships of genes on chromosomes mayreflect both functional significance and evolutionary history.For example, genes of related function are often grouped intoclusters (1-3). In many cases, such arrangements may simplyreflect the mechanisms of duplication by which these genesarose. However, the potential for clustered genes to becoordinately influenced by shared regulatory elements orchromatin structures suggests that such arrangements may befunctionally significant. The Antennapedia and Bithoraxgene complexes, which have been conserved from Droso-phila to mammals (4, 5), are illustrative of arrangements thatare likely under functional constraint.An unusual example of such clustering is demonstrated by
two genes involved in the development and function of theDrosophila melanogaster visual system. The seven in ab-sentia (sina) gene encodes a nuclear protein required for theproper specification of the R7 photoreceptor cell (ref. 6; forreview, see refs. 7 and 8). In flies mutant for sina, precursorcells that would normally develop into R7 neurons insteaddifferentiate as nonneuronal cone cells. The sina gene islocated within a 9-kilobase (kb) intron ofthe Rh4 gene, whichencodes a photosensitive opsin (9). Interestingly, Rh4 isexpressed exclusively in R7 photoreceptor cells, those cellsthat are missing in sina mutants. Although the intimateassociation of these genes is intriguing in light of their relatedfunctions, the significance of this arrangement is unclear. Forexample, the expression patterns of the two genes are notidentical; Rh4 expression begins in the last stages of eyedevelopment and is restricted to the R7 cell (9), whereas thesina gene is expressed earlier and in a greater range of celltypes (6). Additionally, the sina and Rh4 proteins are struc-turally dissimilar and have rather disparate roles in eyedevelopment and function.
If the structural arrangement of the sina and Rh4 genes isfunctionally relevant, one might expect it to be conserved in
other species. To examine this possibility, we cloned andsequenced the sina and Rh4 homologs from Drosophilavirilis* and examined the physical organization ofthese genesin this species. Our results indicate that although the codingsequences of sina and Rh4 are highly conserved between D.virilis and D. melanogaster, the chromosomal arrangementsof these genes are not.
MATERIALS AND METHODSA A EMBL3 D. virilis genomic library (from M. Scott,Stanford University) was screened at reduced stringencywith a 1.9-kb BamHI-Xho I fragment of D. melanogastersina 2CI cDNA (6). Filters were hybridized at 450C in 5xstandard saline citrate (SSC) (lx SSC is 150 mM NaCl/15mM sodium citrate)/100 mM NaH2PO4, pH 6.8/0.1% poly-vinylpyrrolidone-40/0. 1% bovine serum albumin/0.1%Ficoll/0.1% SDS/sonicated salmon sperm DNA at 100 pg/mland washed at 50°C in 0.2x SSC/0.1% SDS. Eight positiveclones were obtained from 105 plaques screened. A 2.8-kbXho I-HindIII fragment ofphage DNA that hybridized to the2CI cDNA probe was identified by DNA blot analysis. Thisfragment was sonicated and subcloned into M13mplO forsequencing by the dideoxynucleotide chain-terminationmethod (10). Sequences were analyzed by using IntelliGe-netics software.A 2.2-kb EcoRI D. virilis genomic DNA fragment contain-
ing the Rh4 gene was isolated (ref. 11; and M. Fortini,personal communication). The nucleotide sequence of thisfragment was determined as described above. Additionalgenomic DNA from the D. virilis Rh4 region was isolated byscreening the A EMBL3 library with this fragment.
Salivary gland polytene chromosomes were isolated fromD. virilis third instar larvae and hybridized, as described byZuker et al. (12). Probes were generated from 2.8-kb XhoI-HindIII and 2.2-kb EcoRI genomic fragments containingthe sina and Rh4 genes, respectively. The probes werelabeled by nick-translation with biotinylated dUTP and de-tected with streptavidin-conjugated horseradish peroxidase,by using the Detek I-HRP kit (Enzo Diagnostics) and thenstained with diaminobenzidine. Chromosomes were hybrid-ized with each probe individually or both probes simultane-ously.
RESULTS AND DISCUSSIONCloning and Sequencing of D. virUs sina and Rh4. The D.
virilis sina gene was isolated by screening aD. virilis genomiclibrary with a D. melanogaster sina cDNA, as described. InFig. 1 the nucleotide and predicted amino acid sequences ofD. virilis sina are displayed and compared with the D.melanogaster sina gene (6). The two predicted proteins are
*The sequences reported in this paper have been deposited in theGenBank data base (accession nos. M77281 and M77282).
10203
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
M S N K I N P K R R E P T V A A A V A A A T A V V A T N T S S S T G S S A- - - - - - - - - - - - - A - - - G - G - - . G - - - - - - T - - - S - - -
GGNAACACTTCSTCGGCAACACATCNTC TCGTC CAGCTCTTCACTGTCGTC CGC CGGTGGCGGTGATGCGGGCATGTCCGCSCGATTAACATCSCTFTTCGATGCCCCGTTTGCTTCG N T S S A N T S S S S S S S L S S A G G G D A G M S A D L T S L F E C P V C F__-
- A- -\-- - -- -_________-- --T -C -T C-GATTATGTGCTGCCGCCGATTCTGCAATGCTCCAGCGGGCATTTGGTGTGCGTTTCGTGCCGTTCGAAGCTCACATGCTGTCCAACATGCCGCGGCCCATTGGCCAACATACGCAACCTGD Y V L P P I L Q C S S G H L V C V S C R S K L T C C P T C R G P L A N I R N L
__-
GC CATGGAGGAGGTCGCCTC CAATGTGAAGTTTC CGTGCAAGCACTCGGGCTACGGCTGCAC CGCCTCGCTCGTTTACACAGAAAAGAC CGAGCACGAGGAGAC CTGCGAATGC CGGCCAA M E E V A S N V K F P C K H S G Y G C T A S L V Y T E K T E H E E T C E C R Pe- - - K - - - - - - - - - - - - - - - - - - - - -
TACCTATGTCCCTGTCCGGGCGCCTCATGCAAATGGCAGGGACCGCTCGATCTAGTCATGCAGCATCTGATGATGTCCCATAAGAGCATCACAACGTTGCAGGGCGAGGATATCGTCTTTY L C P C P G A S C K W Q G P L D L V Q H L M M S H K S I T T L Q G E D I V F
__-
--------u--u--I-------------a----- L gCTGG CCA CCGACAT CAAT CTGC C CGGCG C CGTTGAC TGGGTTATGATGCAGTC CTG CTTTGGC CATC ATTTCATGC TCGTGC TGGAGAAACAGGAGAAATACGATGGACATC AACAGTTCL A T D I N L P G A V D W V M N Q S C F G H H F M L V L E K Q E K Y D G H Q Q F
__-
TTTGCC ATIGTTCAV ITGATG G KCA EGGAG CGAGGACTTTGTCTATCRGCTGGARLTAGGCAXCGACGCCGC T HACETGGGAGGCA RATACACGAG
F A I V Q L I G S R K E A E N F V Y R L E L N G N R R R L T W E A M P R S I H E__-
GGCGTTGC TCCGCCATACACAATTCGGATTGTCTGGTGTTTGATACATCGATTGCGCAACTGTTTGCCGATAATGGCAATCTGGGCATCAATGTAACCATATCTCTGGTCTAAATAATGG V A S A I H N S D C L V F D T S I A Q L F A D N G N L G I N V T I S L V *
FIG. 1. DNA and deduced amino acid sequences of the sina gene from D. virilis (VIR) and comparison with the protein-coding sequenceof the 0. melanogaster (MEL) sina gene. Dashes indicate positions of nucleotide or amino acid identity; gaps in the sequence are indicated bydots.
identical in their carboxyl-terminal halves. They differ by sixamino acid substitutions and two single amino acid insertion/deletions in their amino-terminal halves; five of these sub-stitutions are conservative changes (Ala/Gly, Ala/Val, Gly/Val, Ser/Thr). The single nonconservative substitution is atposition 121, where a lysine in the D. melanogaster gene isreplaced by a glutamate residue in D. virilis. This level ofconservation over the entire coding region (97% identity) ismuch higher than that observed in previously describedcomparisons between these species of several other genes(typically 60-80%o identity; refs. 13-15). The D. virilis and D.melanogaster lineages are thought to have diverged 60 to 80million years ago, enough time to ensure that only sequencesunder functional constraint will be conserved (16, 17). There-fore, such extensive conservation over the coding region ofthe sina gene may reflect important contributions of nearly allof the amino acid residues to sina function.The D. virilis Rh4 gene was isolated in a previous study of
the Drosophila opsin promoters (ref. 11; and M. Fortini,personal communication). In Fig. 2, the sequence of a 2.2-kbgenomic DNA fragment containing the D. virilis Rh4 gene isaligned with the protein-coding sequence of the D. melano-gaster Rh4 gene (9). Comparison of the conceptually trans-lated Rh4 proteins from these species reveals that they are94% identical and 96% similar, allowing for conservativesubstitutions (Asp/Glu, Ile/Leu, Ile/Val, Ser/Thr). Approx-imately halfofthe amino acid substitutions are clustered nearthe amino and carboxyl termini of the Rh4 protein, whichcorrespond to the predicted extracellular and cytoplasmicdomains, respectively. In addition, the colinearity of the D.virilis and D. melanogaster sequences is disrupted by threeseparate insertion/deletions, resulting in a length differenceof five amino acids between the two proteins.Chromosomal Arrangement of the D. vbirs sisa and Rh4
Genes. To determine whether the close physical associationof the sina and Rh4 genes found in D. melanogaster isconserved in D. virilis, we first screened a replica lift of theD. virilis library with a probe containing Rh4 sequences. Five
positive clones were identified, none of which cross-hybridized with the sina probe. Restriction mapping of theseclones demonstrated that they define a region of genomicDNA distinct from the D. virilis sina region (data not shown).To determine the cytological location of these genes, D.virilis salivary gland polytene chromosomes were hybridizedwith sequences from the cloned sina and Rh4 regions. Eachprobe hybridized to a distinct location on chromosome 3; theRh4 probe to the 39B-C region, and the sina probe to 32C-D(Fig. 3). This is in contrast to the arrangement of these genesin D. melanogaster, where sina and Rh4 are located togetheron the third chromosome at 73D (6, 9). Taken together, theseresults indicate that sometime after the D. virilis and D.melanogaster ancestral lineages diverged, one ofthese geneswas translocated to a different chromosomal position.We reasoned that if one gene had translocated while the
other remained in its original location, then the arrangementofgenes near the translocated gene should differ between thetwo species, whereas the order of genes surrounding thestable gene might be conserved. Previously, several tran-scription units near the D. melanogaster Rh4/sina regionwere identified (6). To determine which gene moved, wetested the D. virilis sina and Rh4 regions for the presence orabsence of these sequences by DNA blot analysis. Theresults of this experiment are summarized in Fig. 4. Overall,we found that the arrangement ofgenes flanking the Rh4/sinaregion ofD. melanogaster is highly similar to the sina regionof D. virilis, whereas no such similarity to the D. virilis Rh4region was detected. Specifically, of four probes generatedfrom the D. melanogaster sina/Rh4 region, three hybridizedto blot transfers of cloned genomic DNA from the D. virilissina region in the same linear order in which they appear onthe D. melanogaster chromosome (Fig. 4). In contrast, noneof the probes hybridized to the D. virilis Rh4 region (data notshown). The simplest interpretation of these data is that theRh4 gene moved into another chromosomal site in one of thetwo lineages, whereas the sina gene remained at its originalposition. The Rh4 gene may have been translocated either
VIRVIRVIRVIR
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
MELVIRVIRMEL
VIRVIRVIR
10204 Genetics: Neufeld et al.
Genetics: Neufeld et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10205
VIR GTTTTGCCCAAGAGTATTGTATACTCACGTAGCCCCCCCCCCACAAAT2CAAATTAGA-CATTTGCCTGCAAAATCAATTATT6TGTGGCTTTTTAAGCCAAGTTGCTGTGCATTTAAGCC120VIR AACTGCACAGTTGGCAAAAAAACGAATTTGGCGGCACGCAAGCCCACAACCAAATTGAATTAGTCGCTGCCAAAGGTCTGTCCAACGTATAAAACACGCTGTGCGTGGGGCAGACCACAA 240
MEL -G.. ... C--T-------C-----T---.. --GCCA--T--C-----A--G--6AG---T--------A--CT-A-----CVIR GCCAAAAGTTGGACAGGCGGCTCGTAAATAATGGACATCGCGGGTTC3GCTGTGCAATGCCAGCGAGGTCCTGTGCTGCGACCGGAGGCACGCGTCTCGGGCAACGGTGATCTGCAGTTT360VIR M D I A G S L C N A S E G P V L R P E A R V S G N G D L Q F 30MEL - E . . . P - - - - - - . - P - - - - - - S - - - - - - - -
MEL C-C--A-----------G-----------T--AT-C--T---------------------C----A-----.C--G--CC-----C--C -----6-----C.--------A-----C------VIR TTGGGCTGGAATGTGCCACCGGATCAGATACAGCATATACCGGAGCACTGGCTGACCCAGTTGGAGCCGCCAGCCTCGATGCATTATATGCTTGGCGTGTTCTACATCTTTCTGTTCTGC 480VIR L G W N V P P D Q I Q H I P E H W L T Q L E P P A S M H Y M L G V F Y I F L F C 70MEL - - - - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - -
MEL --C-----A--G--T--T--------C------------AGC---------T--C--A-G-----------------C--G--------------C-----TC-G--C------------VIR GCTTCGAC CGTTGGCAACGGCATGGTTATCTGGATCTTCTCGACATCCAAGGCGTTGCCACACCATCCAATATGTTTGTACTGAATCTGGCCGTGTTCGACTTTATAATGTGCCTCAAG 600VIR A S T V G N G M V I W I F S T S K A L R T P S N M F V L N L A V F D F I M C L K 110MEL - - - - - - - - - - - - - - - - - S - - - - - - - - - - - - - - - - L - - - - -
MEL -------------------------------- A--A--C--CC-------T---T-G-----G--A--C---T----T--C--C--------T-----T--T--------G--C------VIR GCGCCGATCTTCATCTACAACAGCTTCCATCGCGGTTTTGCTTTGGGCAACACCGGCTGCCAAATCTTTGCCGCCATCGGTTCGTATTCGGGCATTGGGGCCGGTATGACCAATGCGGCC 720VIR A P I F I Y N S F H R G F A L G N T G C Q I F A A I G S Y S G I 6 A G M T N A A 150MEL - - - - - - - - - - - - - - - - - - W----- S - - - - - - - - - - - - - - -
MEL --A--A--------ATA---------C--C-----------C------------------------G-G---------A-A-----C---T-G--------A--G--------CC-----VIR ATCGGCTACGATCGCCTCAATGTGATAACGAAGCCCATGAATCGCAACATGACCTTCACCAAGGCGATCATAATGAATGTTATCATATGGCTTTACTGCACGCCATGGGTTGTTTTGCCG 840VIR I G Y D R L N V I T K P M N R N M T F T K A I I M N V I I W L Y C T P W V V L P 190MEL - - - - - Y - - - - - - - - - - - - - - - - V - - - - - - - - - - - - -
MEL GTGAG-9.0 KB-TCCAG
MEL --------------------A--CA----- ..------C--C--G--C---T-C--C--C--T--TT----C--------C--C--GT-------G--------C-----T--C-----CVIR CTAACCCAGTTCTGGGATCGGTTTGTGCCCGAGGGCTATCTGACATCGTGCACATTTGATTACCTAACGGATAACTTTGATACGC6CCTGTTTGTTGGCACCATTTTCTTCTTTAGCTTT 960VIR L T Q F W D R F V P E G Y L T S C T F D Y L T D N F D T R L F V G T I F F F S F 230MEL - - - - - - - - - - - - - - - - - S - - - - S - - - - - - - - - - - - - - - - -
MEL -----T--------G------C--T--------TCG-----C--G--C-----C-----------A-----C--A--G-----G-----G--A-----C--G--G---C-------C---VIR GTGTGCCCCACGCTAATGATCATCTACTACTACAGC1CAGATAGTTGGACAT6TTTTCAGCCAC6AGAA6CTCTGCGCGA6CAAGCCAAAAAGATGAATGTCGAATC6TT6CGCTC6AAT1080VIR V C P T L M I I Y Y Y S Q I V G H V F S H E K A L R E Q A K K M N V E S L R S N 270MEL - - - - - - - L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
MEL --------G--------G--G--G--------G--T--G-----G--T--C-----C-----C-----------6-----------C-----C--A-----------C--G--A------VIR GTGGACAAAAGCAAGGATACCGCCGAGATACGCATCGCAAAGGCAGCCATTACCATATGCTTTCTGTTCTTCGTATCGTGGACGCCGTACGGTGTCATGTCGCTGATTGGAGCTTTCGGG 1200VIR V D K S K D T A E I R I A K A A I T I C F L F F V S W T P Y G V M S L I G A F G 310MEL - - - - - E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
MEL --T------C----T-----A-----.C--G-----C--------------.C-----------G-----A--C--A--C--C--------A-----C.-----A------T----GC----GVIR GACAAGAGTTTGCTAACACCTGGAGCTACCATGATACCGGCCTGCACCTGTAAACTGGTGGCCTGCATCGATCCGTTTGTGTATGCCATCAGTCATCCCAGGTACCGCATGGAATTGCAA 1320VIR D K S L L T P G A T M I P A C T C K L V A C I D P F V Y A I S H P R Y R M E L Q 350MEL -. . . . . . . . . . . . . . . . .- - -
MEL --------T----------GAG-CA-C--A---T-TG6G---AT-TCT------CA----.-. .----C--C.--------G----G--T--C--T--A---VIR AAGCGCTGCCCCTGGCTGGCCATTGATGAGAAGGCGC1CCGAGTCCAGCTCGGCG6CGTCCACAACAACAACACAGAGCAACAGCAAACAACTGCAGCCTAGGCAAAGACTAGAATGAAC1440VIR K R C P W L A I D E K A P E S S S A A S T T T T Q E Q Q Q T T A A * 383MEL - - - - - - G V N - - S G - I - - - Q - . - - - - - - - - - - - -*
FIG. 2. Comparison of the D. virilis and D. melanogaster Rh4 genes. Position of the 9.0-kb intron in the D. melanogaster Rh4 gene isindicated. The putative poly(dA) tail and potential polyadenylylation signal sequence in the 3' untranslated region ofD. virilis Rh4 are underlined.A 5-base pair (bp) direct repeat sequence located adjacent to the poly(dA) sequence and in the 5' untranslated region is overlined.
away from the Rh4/sina region in the D. virilis lineage or into The D. virs Rh4 Gene May Represent a Retropoon. Bythe sina region in the D. melanogaster lineage. what mechanism might the Rh4 gene have moved to a new
,,>~~ ~ ~ ~ N iZ'f*4t4I.| Ij R2ih4'4~~~~h
32DC '398C
Vd]r * ' t
sina
FIG. 3. In situ hybridization of sina and Rh4 sequences to D. virilis salivary gland polytene chromosomes. Probes were generated from 2.1-kbXho I-HindRI (see Fig. 4) and 2.2-kb EcoRI D. virilis genomic DNA fragments containing the sina and Rh4 coding regions, respectively.Hybridization to the sina probe can be detected in the 32C-D region, and hybridization to the Rh4 probe can be detected at 39B-C, as indicatedby arrowheads. (x 1600.) (Insets) Regions of hybridization at increased magnification. (x3000.)
Proc. Natl. Acad. Sci. USA 88 (1991)
(.R:4\1i.........:.h
..
F I ..-..
I~~- - _ . fJl/u-~~~~~~~~~~~- xki/-
In.)
'|a 'A .. .,|lm
FIG. 4. Summary of genomic DNA blot experiments. DNA probes generated from four transcripts in the D. melanogaster sina/Rh4 region(6) were hybridized to DNA blot transfers of cloned D. virilis genomic DNA from the Rh4 or sina regions. (a) Restriction map of the D.melanogaster sina/Rh4 region showing the origins of probes 1-4. Locations of the sina and Rh4 transcripts are indicated. (b) Restriction mapof the D. virilis sina region. Below the map restriction fragments that hybridized to the D. melanogaster probes are indicated. Dashed lines intoneighboring fragments indicate weak hybridization. The 5' end and intron/exon structure of the D. virilis sina transcript have not beendetermined. B, BamHI; R, EcoRI; H, HindIll; S, Sal I.
chromosomal location without disrupting the order of neigh-boring sequences? One mechanism of translocation thatcould account for the above results is suggested by thediscovery of transposed genes that bear the hallmarks ofRNA processing. Such "retroposons," which are thought toresult from the chromosomal integration of DNA copies ofRNA transcripts (18), are usually devoid of introns found inthe original gene and often possess a 3' poly(dA) tail andflanking direct repeats that mark the site of integration.Although many of these processed genes are pseudogenes,several functional retroposons have been reported (19-21);these may reflect either serendipitous integration near anactive promoter or transposition of upstream control ele-ments along with the coding sequences.To investigate whether the translocation of Rh4 occurred
by a retrotransposition mechanism, we examined the se-quence ofthe D. virilis Rh4 gene for signs ofRNA processing.As shown in Fig. 2, the single 9.0-kb intron that disrupts thecoding region of the D. melanogaster Rh4 gene is not presentin D. virilis Rh4. In addition, we found a run of 18 deoxya-denosine residues in the 3' untranslated region ofthe D. virilisgene, preceded by a potential polyadenylylation signal (Fig.2). A 5-bp sequence (CAAAT) that immediately follows thepoly(dA) sequence is repeated twice in the 5' region of Rh4and could represent a direct repeat formed by an insertionevent. These data are consistent with the transposition of theRh4 gene from its location in the sina region into a newchromosomal site by means of an RNA intermediate. Thisinterpretation implies that the D. melanogaster arrangementrepresents the original configuration of the Rh4 and sinagenes and that the transposition event took place in thelineage leading to D. virilis.The extensive sequence similarity of the D. melanogaster
and D. virilis Rh4 upstream control elements (11) suggeststhat, if this model is correct, these sequences were trans-posed along with the coding region; this could have resultedfrom use of an aberrant transcriptional start site by the Rh4promoter or been due to read-through of an upstream tran-script. In this light, the small size ofthe Rh4 regulatory regionis noted; a 190-bp sequence of DNA from the D. virilis Rh4promoter is sufficient to generate an R7-specific expressionpattern in D. melanogaster (11). This 190-bp sequence isentirely contained within the region bound by the CAAATrepeats. Therefore, a transcript beginning upstream of thenormal Rh4 start site could contain the Rh4 coding region andall necessary regulatory sequences in <2 kb.
If the position of Rh4 in D. virilis reflects a retrotranspo-sition event, the original copy of the Rh4 gene would haveremained at the sina locus. In blot analyses ofcloned genomicDNA from the D. virilis sina region, we found no evidence of
Rh4 sequences in this region, even under hybridizationconditions of very low stringency (data not shown). How-ever, if the transposition event occurred soon after theseparation of the two lineages, the original Rh4 sequencewould have had ample time to diverge beyond the limits ofdetection, as only one copy of Rh4 would have been underselective constraint.
CONCLUDING REMARKSThe observation of gene clusters that have been conservedfrom Drosophila to mammals (4, 5) suggests that the physicalrelationships of genes on chromosomes may reflect an un-derlying functional significance. Several examples of geneslocated within introns have now been reported (22-24).Although the relevance of such arrangements is not known,the potential for unique regulatory interactions involvingpromoter occlusion or interference with translation or proc-essing by anti-sense transcripts has been recognized (25, 26).In the case of sina and Rh4, the functional relationship ofthese genes implied that their physical arrangement observedin D. melanogaster might be significant. However, we findthat this arrangement is not conserved in D. virilis, indicatingthat it is not required for the normal regulation ofthese genes.Rather, we suggest the arrangement ofD. melanogaster sinaand Rh4 may be coincidental or reflect a previous signifi-cance in an ancestral species.We thank Todd Laverty for performing in situ hybridizations to
polytene chromosomes and members of the Rubin laboratory forhelpful discussions and comments on the manuscript. This work wassupported, in part, by a National Science Foundation PredoctoralFellowship (T.P.N.) and a Helen Hay Whitney Postdoctoral Fellow-ship (R.W.C.).1. Efstratiadis, A., Posakony, J. W., Maniatis, T., Lawn, R. M.,
O'Connell, C., Spritz, R. A., DeRiel, J. K., Forget, B. G.,Weissman, S. M., Slightom, J. L., Blechl, A. E., Smithies, O.,Baralle, F. E., Shoulders, C. C. & Proudfoot, N. J. (1980) Cell21, 653-668.
2. Garcia-Bellido, A. (1979) Genetics 91, 491-520.3. Thomashow, L. S., Milhausen, M., Rutter, W. J. & Agabian,
N. (1983) Cell 32, 35-43.4. Graham, A., Papalopulu, N. & Krumlauf, R. (1989) Cell 57,
367-378.5. Duboule, D. & Dolld, P. (1989) EMBO J. 8, 1497-1505.6. Carthew, R. W. & Rubin, G. M. (1990) Cell 63, 561-577.7. Tomlinson, A. (1988) Development 104, 183-193.8. Banerjee, U. & Zipursky, S. L. (1990) Neuron 4, 177-187.9. Montell, C., Jones, K., Zuker, C. & Rubin, G. (1987) J.
Neurosci. 7, 1558-1566.10. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl.
Acad. Sci. USA 74, 5463-5467.11. Fortini, M. E. & Rubin, G. M. (1990) Genes Dev. 4, 444-463.
10206 Genetics: Neufeld et al.
Genetics: Neufeld et al.
12. Zuker, C. S., Cowman, A. F. & Rubin, G. M. (1985) Cell 40,851-858.
13. Heberlein, U. & Rubin, G. M. (1990) Proc. Nat!. Acad. Sci.USA 87, 5916-5920.
14. Michael, W. M., Bowtell, D. D. L. & Rubin, G. M. (1990)Proc. Nat!. Acad. Sci. USA 87, 5351-5353.
15. Kassis, J. A., Poole, S. J., Wright, D. K. & O'Farrell, P. H.(1986) EMBO J. 5, 3583-3589.
16. Beverly, S. M. & Wilson, A. C. (1984) J. Mol. Evol. 21, 1-13.17. Throckmorton, L. H. (1975) in Handbook of Genetics, ed.
King, R. C. (Plenum, New York), Vol. 3, pp. 421-469.18. Sharp, P. A. (1983) Nature (London) 301, 471-472.19. Stein, J. P., Munjaal, R. P., Lagace, L., Lai, E. C., O'Malley,
B. W. & Means, A. R. (1983) Proc. Nat!. Acad. Sci. USA 80,6485-6489.
Proc. Nat!. Acad. Sci. USA 88 (1991) 10207
20. Boer, R. H., Adra, C. N., Lau, Y.-F. & McBurney, M. W.(1987) Mol. Cell. Biol. 7, 3107-3112.
21. Ashworth, A., Skene, B., Swift, S. & Lovell-Badge, R. (1990)EMBO J. 9, 1529-1534.
22. Henikoff, S., Keene, M. A., Fechtel, K. & Fristrom, J. W.(1986) Cell 44, 33-42.
23. Chen, C., Malone, T., Beckendorf, S. K. & Davis, R. L. (1987)Nature (London) 329, 721-724.
24. Wallace, R. M., Marchuk, D. A., Anderson, L. B., Letcher,R., Odeh, H. M., Saulino, A. M., Fountain, J. W., Brereton,A., Nicholson, J., Mitchell, A. L., Brownstein, B. H. & Col-lins, F. S. (1990) Science 249, 181-186.
25. Nepveu, A. & Marcu, K. B. (1986) EMBO J. 5, 2859-2865.26. O'Hare, K. (1986) Trends Genet. 2, 33.
