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Homology with Saccharomyces cerevls ae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of f. o.rked occurs at the level of RNA stabd ty

Andrew Mitchelson, Martine Simonelig, 1 Carol Williams, and Kevin O'Hare 2 Department of Biochemistry, Imperial College of Science Technology and Medicine, London SW7 2AZ, UK

The suppressor of forked [su(f)] locus of Drosophila melanogaster encodes at least one cell-autonomous vital function. Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted. Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs. We have cloned the su(f) gene by P-element transposon tagging. Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region. P-element- mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype. The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females. The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability. This suggests that phenotypic modification by su(f) occurs at the level of RNA stability. [Key Words: Drosophila; modifier gene; transposable element; suppression] Received October 12, 1992; accepted November 23, 1992.

Mutations at the suppressor of forked locus [su(f)] lead to can be detected, and the flies have defective bristles. alterations of the phenotypes associated with mutations Lack of either su(Hw) or su(f) products results in sup- at unlinked genes. Phenotypes may be made more mu- pression of the mutant bristle phenotype and accumula- tant {enhanced) or less mutant {suppressed). The su(f) tion of apparently wild-type f RNAs. gene is one of several Drosophila modifier genes, includ- Genetic studies have shown that the different suppres- ing the suppressor of Hairy wing [su(Hw)], suppressor of sor genes act independently, suggesting that they inter- sable [su(s)], suppressor of white-apricot [su(w~)], and vene at different stages in the production of an mRNA suppressor of purple [su(pr)] loci. Mutations of these (Rutledge et al. 1988}. Steps where suppressor genes modifier genes affect the phenotypes of at least 35 mu- might act include transcription, splicing, polyadenyla- tant alleles of 17 different loci (Rutledge et al. 1988; tion, and RNA stability. Several suppressor genes have Lindsley and Zimm 1992). been cloned, and their putative gene products are consis- Alleles that respond to these trans-acting modifiers tent with this hypothesis. The su(Hw) gene was sug- have retroviral-like transposable elements inserted in gested to encode an activator of gypsy transcription noncoding regions of the genes. The insertions either (Parkhurst and Corces 19861, and the gene product has eliminate or alter the pattern of transcription, resulting multiple zinc finger motifs like those found in other in a mutant phenotype. Mutation of the modifier gene DNA-binding proteins {Parkhurst et al. 1988). Analysis appears to modulate further the pattern of RNA pro- of su(w ~) showed that it affected its own RNA splicing duced and so suppress or enhance the mutant phenotype. pattern {Zachar et al. 1987), and the gene product has For example, in forked 1 (fl) a gypsy element is inserted arginine/serine-rich motifs similar to those found in within an intron of the forked (f) gene (Parkhurst and other RNA splicing proteins (Li and Bingham 1991). The Corces 1985; McLachlan 1986). No stable f transcripts su(s) gene also appears to affect splicing (Fridell et al. 1990), perhaps at the level of precursor RNA stability (Geyer et al. 1991). It encodes a nuclear protein similar to 1Present address: Dynamique du genome et evolution, Institut Jaques Monod, Universite Paris 7, 75005 Paris, France. RNA-binding proteins with ribonucleoprotein (RNP) 2Correspondingauthor. motifs (Voelker et al. 1991).

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Mitchelson et al.

Most su(f) alleles are lethal, consistent with there be- in the original mutant stocks was then substantially re- ing an important role for the gene product. Of the viable duced by substituting the autosomes and recombining alleles, four are temperature-sensitive lethals (Dudick et away the distal portion of the X chromosome. Several al. 1974; Russell 1974; J/irgens and Gateff 1979; Wilson independent recombinant sublines were then analyzed 1980). They have similar periods of temperature-sensi- by DNA blotting, and the one with the fewest remaining tive lethality from late first-instar larvae to just after P elements, a subline of su(f) 28, was used as a source of puparium formation. Analysis of somatic clones has DNA to construct genomic libraries in k vectors. Phage shown that the lethal phenotype is cell autonomous. with homology to the P element were purified and sorted The su(f) gene therefore encodes a vital cell-autonomous into groups corresponding to sites where a P element is function, and we presume that this function is related to inserted in this particular su(f) 28 subline. Flanking the phenotypic suppression of some mutations caused by probes were then used in DNA blotting experiments to insertion of transposable elements. We undertook the identify the element associated with the lethal pheno- cloning of the su(f) gene as a vital cell-autonomous gene type of su(f) 28. Figure 1A shows that the B1 group P el- the role of which is likely to be in mRNA production or ement is absent in six independent revertants of su(f) 28. stability. In contrast, hybridization of the same filter to a probe flanking the $7 P element (Fig. 1B) showed that it was present in both mutants and revertants. Analysis of Results DNA from su(f) 27 flies showed that this allele also has an insertion in the region where the B 1 group P element is Cloning of DNA sequences from su(f) inserted (see Fig. 4, below) and that this insertion was The su(f) gene is the most proximal single-copy gene in absent in revertants of su(f) 2z. Subsequent cloning exper- the map of the X chromosome (Schalet and Lefevre iments confirmed that the insertion detected in su(f) 2z is 1973). It is within the euchromatin-heterochromatin a P element (see below). transition region at the base of the X. It has proved dif- Sequences flanking the B 1 group P element were used ficult, or impossible, to clone genes from this region by to screen a Sau3AI partial digest library of wild-type chromosomal walking because there are large blocks of (Canton-S) DNA (Mariani et al. 1985). Figure 2 shows a repeated sequences (Miklos et al. 1984, 1988). We there- restriction map of the 33 kb isolated and the site of in- fore cloned DNA sequences from su(f) by P-element sertion of the P element in su(f) 28. The SalI site 150 bp to transposon tagging. A collection of 110 lethal mutations the left of the insertion site was designated coordinate induced on the X chromosome by PM hybrid dysgenesis 0.0. On the basis of the mapping of breakpoints associ- (M. Simmons, University of Minnesota, St. Paul) was ated with chromosomal rearrangements (L. Kelly, pets. screened for su(f) mutants. Two stocks, 1(1)M897 and comm.; A. Mitchelson, M. Simonelig, C. Williams, and 1(1)MS252, were found to complement a temperature- K. O'Hare, unpubl.), we have drawn the DNA maps so sensitive lethal su(f) mutant at the permissive tempera- that left to right is distal to proximal (- to +) with ture but not at the restrictive temperature. respect to the centromere. DNA blotting experiments To ensure that these stocks had bona fide P-element have revealed the presence of appreciable amounts of insertion mutations of su(f), they were tested for rever- repetitive DNA in the cloned interval (data not shown); sion during hybrid dysgenesis. Both I(1)MS97 and the approximate extent of repetitive sequences is shown 1(1)MS252 gave viable revertants at rates between 3 x in Figure 2. An analysis of the repetitive DNA sequences 10 -3 and 7 x 10 -3 and have been designated su(f) 27 and in the su(f) region will be presented elsewhere (M. Tudor, su(f) 28, respectively. The number of P elements present A. Mitchelson, and K. O'Hare, in prep.).

Figure 1. Identification of the P element responsible for su(f) 28. {A) DNA samples were digested with BamHI and SalI and probed with pB1 .L, a fragment flanking the B1 group P element. In flies heterozygous for su(f) 28, a second band larger than wild type is present; in each revertant line, only the wild-type band is seen. DNA samples: (Lane 1) Canton-S; {lane 2) FM6; (lane 31 su(f)28/FM6; (lane 4} subline 1 of su(f)28/ FM6; (lanes 5-7) three independent viable revertants of subline 1; (lane 8) subline 2 of su(f)28/FM6; (lanes 9-11) three indepen- dent viable revertants of subline 2. (B) The same filter probed with pS7.SR; a fragment flanking the $7 group P element. In each revertant line, the $7 P element is re- tained. Fragment sizes are in kb at right.

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Drosophila modifier gene

where in the genome. We cannot exclude that the flank- ~ = = , I z , , , I R , , , ~ , , , ~ ~ , , I , = J , J J J J , ing repeated sequences have a role in allowing expres- transformed region sion of the gene in its normal location at the euchromatin-heterochromatin boundary. ;,=, ,=,,.xDx xx,.; ,,. II 1 I III II i r i i~ i i i i~ = = H == = J = S HH SS H S H HH H HSHHSH H H Transcription of the su(f) region V I 252.B1 = CSR5 A series of RNA blotting experiments were performed g CSR3 I CSR2 using strand-specific probes to map transcripts in the I CSP,1 I CSP.4 region between + 5.6 and - 2.1. Three RNA species were detected that are transcribed from distal to proximal: a Figure 2. Restriction enzyme site map of the su(f) region. Re- major RNA of 2.6 kb and minor RNAs of 2.9 and 1.3 kb. gions of repetitive DNA are shown as thick lines. The ~, clones CSP.1-CSP.5 are from Canton-S DNA, whereas 252.B1 is from Probes from 0.0 to + 2.1 detected all three RNAs while su(f) 28 flies; the position of the 1.1-kb P-element insertion is probes from +2.1 to +4.3 detected only the 2.9- and marked on this clone. The coordinate system is shown above 2.6-kb RNAs. A profile of transcription of this region the map. Position 0.0 is the Sail site close to the P-element through development is shown in Figure 3A. All three insertion site in su(f)2s; each unit represents 1 kb. The interval RNAs are present at all stages and are most abundant in introduced into flies by P-element-mediated transformation is embryos, early pupae, and adult females. shown. Restriction enzyme site abbreviations: (R) EcoRI; (X) Transcription in four mutants was examined (Fig. 3B). XhoI; (B) BamHI; (H) HindlII; (S) Sail. For two lethal insertion mutants, su(f) 26 and su(f) as, RNA was prepared from adult females heterozygous with the X chromosome balancer FM6, which has a wild- type copy of su(f). In su(f) a6 a Doc transposable element Rescue of su(f) by P-element-mediated transformation is inserted at +3.2 (Driver et al. 1989). No mutant-spe- To confirm that the single-copy region disrupted in the cific RNAs were detectable; the su(f)a6/FM6 sample was P-element insertion mutants was the surf) gene and to indistinguishable from wild-type. In the su(f)aS/FM6 define the limits of the gene, a fragment from -2.1 to sample, there was a mutant-specific RNA of 3.7 kb. We + 4.3 (Fig. 2) was introduced into flies by P-element-me- believe that the 3.7-kb RNA in su(f) 2s is a cotranscript diated transformation. Two independent transformants where the P-element insertion has simply increased the were obtained. The transformants rescued lethal surf) size of the exon at the point of insertion (see below) by alleles for viability and viable alleles for the suppression 1.1 kb. For the viable mutants su(f) 1 and su(f) 14, RNA of f phenotype. We conclude that the fragment from was prepared from homozygous adults. Although these -2.1 to +4.3 includes the structural sequences of su(f) mutants appear to be point mutations, their RNAs did and all of the flanking sequences necessary for wild-type show differences from wild type. In su(f) ~, the 1.3-kb levels of expression. This interval is single copy and transcript is not evident, and there are two new RNAs of shows that the repeated sequences that normally sur- 3.2 and 1.6 kb. In su(f) ~a, the 1.3-kb RNA is less abun- round su(f) are not required for expression at sites else- dant than in wild type.

Figure 3. Analysis of transcription from the su(f) region. (A) A developmental profile of the su(f) transcription unit. The probe used was from 0.0 to + 2.2 and would hybridize to RNAs transcribed from distal to proximal. Poly(A) + RNA samples: {Lane E) 0- to 16-hr embryos; (lane 1) first-instar larvae; (lane 2) second-in- star larvae; (lane 3) third-instar larvae; (lane 4, EP) 7-day pupae; {lane 5, LP) l 1-day pupae; (lane 6) adult females; (lane 7) adult males. {B) A blot of poly(A) + RNA samples from su(D mutants probed as in A. (Lane 1) Canton-S; (lane 21 su(f)l; (lane 3) su(f)Ia; (lane 4) su([)26/ FM6; (lane 5) su(f)aS/FM6. The result of rep- robing the filter with the ras64B probe is shown at the bottom.

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Mitchelson et al.

Analysis of su(f) cDNA clones 2.5 kb. We have determined its sequence and the se- quence of the corresponding genomic interval. Figure 4 Ten independent cDNA clones were isolated from a li- shows the sequence from 100 bp more distal than the brary made using poly(A) + RNA from first- and second- EcoRI site at -0.4 to the PvuII site at + 4.3. Only small instar larvae (Poole et al. 1985) screened with a probe o~ intron sequences are given in their entirety, but the com- the sequences from -2.1 to + 4.3. DNA and RNA blot- plete DNA sequence from the EcoRI site at -3.6 to the ting experiments showed that three of the cDNAs were XboI site at + 6.4 has been entered into the EMBL data from a transcription unit that includes sequences be- base as accession number X62679. The cK22 insert is tween - 1.0 and -9.2 and is transcribed from proximal terminated at its 3' end by a poly(A) sequence, and a to distal (data not shownJ. The remaining seven cDNAs polyadenr signal (AATAAA) is present at its 3' end are from the su(f) transcription unit lying between -0.4 (Fig. 4). This places the 3' end of the transcript corre- and + 4.3. Two additional su(f) cDNAs were isolated us- sponding to this cDNA at +3.4. The sequences of cK13 ing more specific probes: cK25 was isolated from an and cD1 are colinear with cK22 up to the 3' end of exon adult female eDNA library (Poole et al. 1985) using a 4 but then match the genomic sequence up to + 1.7. Both probe from + 0.1 to 0.0, and cD1 was isolated from an cDNAs have 3' poly(A) tracts: >75 bases in cK13 and 38 embryo eDNA library (Brown and Kafatos 1988) using a bases in cD1. The genomic DNA sequence correspond- probe from + 1.2 to + 1.3. ing to the 3' end of cK13 and cD1 is A rich but does not The largest su(f) eDNA clone (cK22) had an insert of include a canonical polyadenylation signal. The insert in

50 .... 100 ~T1AAACGGTT~AAAGTAT~TGTTAACAA~CTT~AC~TTAG~AA~G1ACAGAA~ATGTTT~AGTGG~AC~GCTATkAAAAA1ATTGCTTCTGC1T

22O0 ~CATTATTTACAGATTTA~TACCT T TAAAGTAIGTT~AACCA~ATCT TTTTTAT TTAIAGTTTAT~TCAGIGACC~CTGTACTACACAT~ AAGG..... AC...... AC.... GTTAC...... CAGACATTGA, ~150 CCCACA ..... AGGAACAC~CATAT, G3UkTATG' ' 'CATCAAA' AT ' T GATA K V H T ~ Y N K L L G L P 0 i 0 P T I...... intron 5 : 65 bp ...... J ..... 9c01 250 .... 300 AAATCTTGGGATTTT~TGGTAAGG~CCGCGATGTGAT~GGCGATGACACTA~CGCAGTTACATCCAAT~AGTCGTCGATATCT~GCAGCCGCATTGCA 2250 . . 2300 , [ ..... 9 cK13 CCATCGAAATT TAATACT CCAGGT CTATGT TCAGTACATGAAGT T CGCCCGT CGT GCTGAGGGGATC&AGTCGGCCCGT AGCATAT T TAAGAAAGCTCGG ...... v Y v ~ Y N K F A R R A E G I K S A R S I F K K A R ACTTCT~ ~CGGA~AGC GGCATCC,A~CGT, GCTTTAGGTI~' GCGTAGC~A~GTTCAAC GG. GAAA OG~CTTTCCC GGGs G~ 2350 . . 2400 J ..... > cK25 J..... > cK22 GAGGACGTAAGGTCGCGCTATCACAT TT TCGTGGCCGCTGCITTA~TGGAG TATTATTGCTCCAAGGACAAAGAAAT TGCGTTCCGCAT CT TCGAGC1Go 9 450 .Sail . , 500 E 0 V R S R Y H I F V A k A L M E Y Y C S K D K E ] A F R I F E L AGCGCAAGCAGGAC~C~CGACAAT~TGCAAAGACTAT~A0CAATGTCTT~GGCCAGA~CcTTATcAAAGT~GA~GT~GTAAAA~ATTGGc~TTA MSSARDLIKVDI ...... 9 2450 . . 2500 GAT TGAAGCGTT T I GGTGGCAGTCCAGAGTACGT GATGT GCTACAT TGACT ACCT GTCCCAT CTAAACGAGGACAATAATACGCGCGTCC TGT TTGAACG 9 550 . . . ~0 G L K R F G G S P E V N C Y I D Y L S H L N E 0 M N T it V L F E R TTTGGATGGT~C~CATATTTTTACTTATCGATGAC~GG~TGGGGCATG0AGCGCCTGGTGCGAGCTCAGC~GTGGTGGAGCTGCGGCCCTAT0AC .... intr~ 1 : 61 ~ ...... E W G N E R L V R A ~ 0 V V E L R P Y D 9 . . 2550 Xhol . 9 ~600 GGTACTTAGT TCAGGT GGT T TGTCGCCGCACAAGAGTGTT GAGGTC TGGAAT CGC1 TCCTCGAGT TCCdL~TCTAATAT TGGGCakTCTCTCCAGCATCGT T su(f) 28 . . 650 . su(f) ~7 . 700 V L S S G G L S P H K S V E V W N R F L E F E $ M I 0 D L S S I V ATc~TC~TGGAG~T~ATGAT~CGAGAGG~CCA~CGCGGCC~T~ACG~GTACG~AGTCTTTAC~GTCGCTAGTT~TGTGTTTCCGACAACCG IESWSVMIREAQTRPIHEV SLYES~VNVFPTT 2650 . 2700 AAGGTCGAACGTCGCCGTAGT GCCGT T T TCGAAAACGTGCGTTCT T T TACTACT.T TATT T TAGAAATATGTTATAATAATATT TGT TTAAAGCTTAKAAG~E K V E R R S A V E N ...... mtron 6 : 56 bp ...... L ~,COOT,C,~O.O:,~,,:~ .... O,,OO,O,,O~OC,OCCOO,,:~O~O:O,O~,O.,:,.;,~CO~,O::,,O,:,,O~'T'O"~ ARY~KLYIEN MR RYYERVEKLFORCLVKILNI Pv'ul I 2750 . 2800 GTATGAGGGCAAGGAGACCGCCCAGCTGGT TGACCGT TACAAAT TT CT GGACCTT TACCCCTGCACCAGCACCGAGCTCAAGTCCATAGGAI ATGCTGAG 9 650 . 9 Y E G K E T A 0 L V D R Y K F L D L Y P C T S T E L K S I G Y k E CGACCTCTGGAAGCTCTATCT TACCT ACGTGAAGGAAACCAAGT CGGGT CTAAGTACTCACAAGTAACT GT T ATACCTTA ...... D L W K L Y L T Y V K E T K $ G L S T H K ...... intro~ 2~ : 16/r ~ ...... PSt I . . 3300 GTGCGTAACGGAA...... CACCTCGT T CT GCAGAATGT~,GGCAT TAT ACTGAACAAGGTGG~TGGTGGAGCTCAAAGCC&GAAC 9 1050 . . . 1100 ...... intro~ ? : 449 bp ...... N V G I ] L N K V G G G A ~ S ~ W ..... ACTTATCTCTTTTCTCAGAGAAAAAATGGCCCAGGCTTATGATTTTGCACTG6AGAAAATCGGCATGGATCTkCACTCGTTCAGCATCTGG ...... K M A Q A Y 0 F A L E K I 0 g 0 L H $ F S N 3350 3400 ACCGGAGAGGTCGAAACAGATAGCGAGGCAACGCCACCAT T GCCACGTCCGGATT T CTCCCAGATGAT TCCAT TCAAGCCGCGTCCATGT GCT CATCCTG 9 . . 1150 9 1200 T G E V E T D S E A T P P L P R P D F S Q 14 I P F K P R P C A H P CAGGATTACATATACTT T CT GCGGGGCGTT GAAGCAGTGGGCAACTAT GCAGAGAACCAAAAGAT CACAGCAGT GCGCCGCGTCTATCAAAAGGCTGTAG D Y [ Y F L R G V E A V G N Y A E N 0 K I T A V R R V Y 0 K A V 9 3450 . 3500 CTGCCCATCCACTIGCAGnTGGTGTATTTCCGCAnCCGCCAGCTCTnGCCGCTTTGTGCGCCACCC T OCCTCCACCGAACTcCT TCCGTGGACCGT TCOT 9 1250 . 9 1300 G A H L A G G V F P 0 P P A L A A L C A T L P P P M S F R G P F V TCACACCTA1CGT GGGCATCGAGCAGT T GT CGAAGGACTATA TCG'AT T CGAGCAAAACATAAACCCGAT TAT AT CAGAGAAGATGAGTCT GGAC+CGAIC V T P I V ~ I E C: L ~ K D Y ] k F E 0 N 1 N P ] I S K ~ S L E R S 3550 . 36O0 CAGCGTAGAGCTACTATTCGACAT T T TCATGCGTCTCAATCT TCCGGACT GT GAGTG t CCCTGATTGA-r OTTCAATTAACATTGAACAAT TATOT 9 . 1350 . 9 S V E L L F ] F R L N L P D ...... intron 8 : 57 bp ...... T~GT~A~TGTTATACGCAGA~TTT~CTA~TGTTCTTTCTAAATTCCAAcTTAAAACCAGGGATTACATGAAcGCTCGTCGTGT~GCCA~AGTT~GA K ...... intr~ 3:58 ~ ...... D Y N N A R R V A E L E Bg(|lsu(f) 26 3700 TACT;~GCACCGC~CCG~T GGA~C~CGAATT GTCGCC T~GATCT TC~TCTGGCC~GT CGGTTCACTG~T CGTG~T AC~GTACGT$TA 9 . 1450 . 9 1500 ...... SAPOPNGOMELSPKIFDLAKSVHWIVOT$T ATACCACACAAAGGGTCT~AATCGCAATCT~CCAGCCGTCc~C~CACA~T~ACAAAAGAGGAGGT~AG~AA~TGGAGCTGTGGAAGCGCTTTATCACGY~KGLNR~L~A~TLTKEEVK~ELWKRF~T 9 3~0 . .Pst] 3800

CGGGGGTGCAGCACAGTGTTACGGCCGTTCCACCGCGCCGGC~CGACTG~T~CC1G~GG~C~T~TAGT~C~CGAACT~CA~CC~CTGTGCCGCC 1550 1~0 TGVQHSVTAVPPRRRRLLPGGDDSDDELOT VPP T~T~G~GTCT~TCCCTTGC~CACTGAAGATACGGCCCTAGT~CCGTCGTGTCATGTTCGCCA~GGAGCAATGTTTGCTGGTGCTCACCCACCACC YEKSNPLRTEOTALVTRRVNFATEOCLLVLTHH - ~50 . . 3~0 CT~CCAC~TATATATCGC~TG~T~AGCTAAAGCGTTTCGC~GTCT~TTAAGAAAC~TTAA~AAAAAAC~TTTTTA~TTTTTT~TTTC~TT~ 9 . 1650 . . 9 1700 5HDIYRLRGLKRFAKSN* ~G~AGTGTGGCA~A~GC~TC~C~TTCCTGGACA~CAGTG~T~G~GTGCTCACCG~AAAGG~T~CGTACTAGTGTTGAA~ATTTCAC~TATT~T PAVWHOASQFLDTSARVLTEKGVRTSVENISPIL 9 3~0 . . 4000 P k V W H Q k S 0 F L 0 T S A R V L T E K G ...... intrm 4:2~ ~ ...... TTTAC~GAAA~GAAATCC~TAJ~GC~GCCATATT~TTAGGTTTAGT1TT~TT~GAAGCTGCTTGGGAATTT~CCTTCAT~CTTTGGCATCACC c,22 ..... ,I 1~0 9 4050 .... 4100 CTGCG~TCCTGTAGT~AATCAAATAGAGTGGGTTATGGCCTTTG~TGGTGGTGGG~AAAG~T~AAAA~CAAA~ACG~ATC~A~T~TAG1...... GGTTG~AGACA~TAG~GTTGCA~TGAA~G~TAGCAT~A~U~T~TGT~AT1~TCT1~TGT~GTA~A~ACTAACC~T~CATC~A~TTAAA~TT CVPVVNQIEWVMAFAWWWAKVKTKNE C01, CK13 .... ,I .... 4150 .... 4200 9 1950 .... 2000 ~GTGTA~ATTTGTTGT~ATAA~TAT~CAA~A~AAAGT~CA~TTTT~GATAcTA~AT~TATTTTTTAATTTAGGC~AA~A~GTTATACAT~CT~ ...... ATTTACATOTTTAATTACTTATTAAAACATTGCAGGACGTCCAGGCGGCCAAGATTTTTGCGGACGAOTG cKI ..... >I ...... DVOAAKIFAOEC 9 4250 , . . 43~ AC~TAGTATAT~T~TATAC~TA~ATT/~AATCT~TATAATGGTATA~T~GTA~ATGTTTA~AT~TGAATTT~TGCAT~TTTAAAGTTA~G .... 2050 .... ~100 T~T~TATACTGGAA~GGTCTATAAACGGCG~GCTA~CC~AATGCG~GCTATATTTTGCCTATGCGGAC~T~AA~AAGGAcG~CTCAA~TAC~A~ 9 9 9 P~[[ A N I L E R S I N G V L N R N A L L Y F A Y A 0 F E E G R L K Y E AGTAGAATGTAGACCTAG~AGCACTCCTCCATTACCTCAGCTG Figure 4. Nucleotide sequence of the su(f) locus. Coordinate O0 corresponds to the SaII site at position 471. The positions of the 5' ends of eDNA clones cD1, cK13, and cK25 and the 3' ends of cD1, cK13, cK22, and cK1 are shown. Predicted protein sequences are shown using the single-letter amino acid code below the corresponding DNA sequence. For clarity, where the two are identical, only one is given. The P-element insertion sites in su([y (nucleotide 607} and su(f)~ (nucleotide 674), and the Doc insertion site in su(f) ~ (nucleotide 3645) are underlined. Also underlined is the probable polyadenylation signal (AATAAA) for the 2.6-kb RNA (nucleotide 3922).

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Drosophila modifier gene cK1 has an extra 261 bp downstream from the 3' end of less homologous overall yet include this motif, but no the insert in cK22 that matches the sequence of the ge- striking matches were found. In comparison with the nome (see Fig. 4). This eDNA does not have a poly(A) RNA14 gene product, the 84-kD su(f) protein has a car- tail, and there is no polyadenylation signal close to its 3' boxy-terminal extension of 130 amino acids, 17 of which end nor in the 3'-flanking genomic sequence. We con- are prolines, including runs of 2 and 3 consecutive resi- elude that polyadenylation can occur at + 1.7 for the dues. Similar proline-rich carboxy-terminal regions have 1.3-kb RNA, at +3.4 for the 2.6-kb RNA, and at - +3.7 been described as an activation domain in some tran- for the 2.9-kb RNA. Thus, cK22 appears to be a near scriptional regulators (Mermod et al. 1989) and in complete eDNA clone of the major 2.6-kb RNA, cD1 a poly(A)-binding proteins (Burd et al. 1991 }. The carboxyl complete eDNA clone of the 1.3-kb RNA, and cK1 a termini of poly(A)-binding proteins from different organ- partial eDNA clone of the 2.9-kb RNA. There were no isms are proline rich but are not otherwise homologous. other differences owing to different intron-exon bound- The protein encoded by the 1.3-kb RNA would have aries between these clones and the remaining four eDNA the same 313 amino-terminal amino acids as the 84-kD clones that were effectively internal subfragments of protein. The reading flame continues to be open for cK22. translation to the position where cK13 and cD1 are poly- The 5' ends of cD1, cK13, and cK25 map upstream of adenylated, so the protein would have 37 additional the 5' end of cK22 (Fig. 4). We conclude that the tran- amino acids encoded by the genome and then a polyl- scription start site for su(f) is close to the EcoRI site at ysine tract encoded by the 3' poly(A). These 37 amino -0.4. The different positions that we have assigned for acids have no good matches in data bases. Neither the 37 the 3' ends of the three different sizes of su(f) RNA are residues nor the corresponding region of the 84-kD su(f) compatible with the 5' end of all three RNAs mapping to protein are very similar to RNA14 (Fig. 6). the same position.

Discussion Predicted protein products of su(f) The analysis of RNAs and cDNAs suggests that three su(f) RNAs encode two proteins (Fig. 5). The 2.6- and Cloning the most proximal gene on the X chromosome 2.9-kb RNAs would encode the same 84-kD protein of Drosophila melanogaster whose amino acid sequence is shown beneath the DNA We have characterized and cloned two lethal P-element sequence in Figure 4. Nucleic acid and protein data bases insertion alleles of su(f): su(f) 2z and su(f) 28. Using the were compared with the sequence of the 84-kD su(f) pro- sequences flanking the P elements as probes, we have tein. An extensive homology was found with the RNA 14 isolated 33 kb of wild-type sequences. The euchromatin- gene product from Saccharomyces cerevisiae (Minvielle- heterochromatin boundary region of the X has many dif- Sebastia et al. 1991). The match (Fig. 6) is between the ferent repeated sequences (Miklos et al. 1984, 1988) that entire length of RNA14 and the first 600 amino acids of interfere with gene cloning by chromosomal walking. the 84-kD su(f) protein. The overall homology is 26% The inserts of every recombinant phage that we have identity and 47% similarity. The most conserved region isolated include repeated sequences. However, there are of 10 amino acids has 7 identical and 3 similar residues a number of single-copy regions embedded within the [positions 430-439 of the su(f) protein[. This was used to repeats, one of which includes the sites of insertion of search protein data bases for other proteins that might be the P elements. Transformation of flies with this inter- val has shown that su(f) is contained in a 6.4-kb region. This region hybridizes to larval salivary gland polytene 5 p chromosomes at the cytological location of the su(f) gene at the very base of the X chromosome (Yamamoto et al.

2.9~ 1990). The collection of EcoRI fragments generated by microdissection of this region of the X chromosome (Miklos et al. 1988) includes many of those from the 2.6 kb ~j interval that we have cloned (A. Mitchelson, G.L.G. Miklos, and K. O'Hare, unpubl.).

1.3 kb

R RR X B X Suppressor genes may have roles in I Ill I I II ~ I mRNA metabolism H S H H H Modification of the phenotypes of insertion mutants is not restricted to Drosophila melanogaster. In the yeast 0 +5 S. cerevisiae, mutations at SPT genes suppress the phe- Figure 5. Structure of su(f) RNAs. The deduced intron-exon notypes of mutations due to insertion of Ty and/or Ty map for the three su(f) RNAs is shown above a restriction en- solo long terminal repeats (LTRs) {Winston et al. 1984; zyme map of the locus. The protein-coding regions are filled in. Fassler and Winston 1988). In the mouse, dilute suppres-

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Mitchelson et al.

1 NSSARDLIKVDIE~ER ...... LVRAOOWELRPYDIESWSVNIREAOTR-PIHEVRSLYESLVNVFPTTARYUKLYIENENRSRYYERVEKLFQ su(f) I.-.11 ...... I- :1 ..... m:l I: :m. :1 .... ll.:ll: I.II .. I.I :. m:. :1 II1::. 1 ~SSTTP~LLY~ADKVAE~S~N~HG~ELRLRER~KDNPT~LSYF~L~YLET~ESYAK~REVYE~FHNTFPFYSPAWTLQLKGELARDEFETVEK[LA RNA14

91RCL~K~L~N~DLWKLYLTY~KETKSGL~THKEKNA~AY~FALEK~GN-DLHSFS~WQ~Y~YFLRGVEAVGNYAENQK~TAVRRVY~KAWT~V~ su(f) 9II I ::'11" I1-1 ...... I'l "1 ...... I ..... I1:1":1: II ...... I-I:1- :1 .I-I :''1: 9 101 ~CL~GKLENN~L~LWSTYLDY~RRK~NL~TGG~EARA~KAF~LW4QKCA~FEPKSS~FWNEYLNFLE~K~FNK1~E~R~D~LREFYKKNLCVpFDN RNA14

184 IEOLWKDYIAFEONINPIISEKNSLERSKDYNNARRVAKELEYHTKGLNRNLPAVPPTLTKE...... EVKQVELWKRFITYEKSNPLRTEDTALV su(f) :1.:1-I.-:11=11 .... I: II :11.11.:-I: I.I1.1. I -I .I .... I=::l .:i.=l:.l-I ...... 201 LEK~'E~/NRYT~/E~E]NSLTARKF~GEL~AEYNKARSLYQEWL~VTNG~K~A~NLRTANKKN~p~GTSDS~LQ~WLNW|KWERE~KLNL~E~D~L RNA14

274 TRRVI4FATEQCLLVLTHHPAV1WHQASQFL-DTSAR...... VLTEKGOVQAAKIFADE--C ...... ANILERSINGVLNRNALLYFAYADFEEGR su(f) 9.1: :--I .... :.:m,: I:: :.m.I .m ...m ..... I I .i.=:: ..... I ...... I=.- 300 ~R~Y~YKQG~Y~IF~AE~WYDYS~YISENSDR~LYTALLAN~SPSLTFKLSE~`CYELD~DSE~VS~CFDKCT~TLLS~YKK~AS~SGED~ RNA14 ..... I. I.: I: I.m .:.: I .... C terminus of protein encoded by 1.] kb RNA VRTSVENISP|LCVPWNQ]E~/ ...... NAFA~AKVKTKNE

]55 LKYEKVHTNYNKLLQLPD~PTLVYV~YNKFARRAEG~KSARS|FKKAREDVRSRYH~|FVAAAL~EYYCSKDKE|AFR~FELGLKRFGGSPEYVgCY~D su(f) 9Im ..11 9 9m:l:. ml. :1 .l:..im.:l m.I. I I::l..I.:l: .I ..im:::lllilm .... i: i:1 398 TEYE...... •ELLYK•REKLTF•FC•Y•NT•KR•••LSAART•F•KCRKLKR•LTH•vYVENAYLEFQNQ•DYKTAFKVLELGLKYF•NDGVY•NKYL• RNA14

454 YL•HLNE•NNTR•LFE•R•LSSGGLSPHKS•EVWNRFLEFE•N[G•LSS•VKVERRRSAVFENLKEYEGKETA•LVDRYKFLDLYPCTSTELKS•GYAEN su(f) Figure 6. Alignment of 84-kD su(f) pro- :1 ml.m ..... iII.m..:l.. I I ...... II.:l:l ..... I:1 .IN ..... I .... II ...... ml. : i.[: tein and RNA14 of S. cerevisiae. Vertical 492 FL•FLNKD•Q•KTLFET•VEK•••LTQLK••E•YKKN•SYESKFG•L•NvYSLEKR-••FFERFPQE•L•EV•-FTSRYQ•Q•••L•KKLELTY••Y•EE RNA14 lines indicate identical residues; colons in- dicate chemically similar residues; periods ******* 9 t w t v,w ft. 9 - show similar-sized residues. The cluster- ...... I::: m: : ...... m...... I.: ing of prolines at the carboxyl terminus of 584 EDSYFSSGNC4)GHHGSYNNSSSDRKRLNEETGNNGNFSNKKFKRRLRASNRGS 6]6 RNA14 the predicted su(f) protein is shown by as- terisks (*). , .-. .. . . **. , 9 ** 9 646 RLNL•DSAP•PNGDNELsPK[FDLAK••HW•VDTSTYTGV•H••TA••PRRRRLLpG•DDSDDEL•TAV••SHD•YRLR•LKRFAK•N 733 su(f)

sor affects the phenotype of a mutation caused by inser- like RNA14, the RNA15 gene product does have RNP tion of an endogenous retrovirns (Jenkins et al. 1981). motifs and is presumably an RNA-binding protein, yet Mutation at suppressor loci leads to different RNAs be- overexpression of RNA 14 complements RNA15 mutants ing produced from the insertion mutants, suggesting that {Minvielle-Sebastia et al. 19911. This observation and the suppressor genes have roles in mRNA production or sta- similarity between the carboxyl termini of PABPs and bility. This indirect genetic approach may complement the 84-kD su(f) protein leads us to suggest that the 84-kD more direct biochemical approaches to identifying and su(f) protein binds to RNA. Alternatively, it may not studying important functions in gene expression. Anal- bind RNA directly but may interact with an RNA-bind- ysis and cloning of Drosophila suppressor genes have ing protein. shown that su(w ~) has an arginine/serine-rich motif A second su(f) protein may be encoded by a 1.3-kb found in RNA splicing proteins (Li and Bingham 1991) RNA from the 5' half of the gene. There is no stop codon while su(s) has an RNP motif found in RNA-binding for the open reading frame in the 1.3-kb RNA. The two proteins (Voelker et al. 1991). We show here that su(f) independent cDNAs [cK13 from a larval library in k and encodes a protein with no previously described motifs, cD 1 from an embryonic library in a plasmid vector) have which is similar to a protein with a role in mRNA sta- exactly the same 3' end in intron 4. The length of poly{A) bility in yeast (Minvielle-Sebastia et al. 1991). at the 3' end of these cDNA clones is not consistent with artifactual generation by oligoIdT) priming at an internal The role of su(f}: predicted protein products A-rich region on a precursor RNA. The sequence of these cDNAs suggests that if the 1.3-kb RNA is translated, the The major product of su(f) is an 84-kD protein encoded protein produced would have polylysine as its carboxyl by 2.6- and 2.9-kb RNAs, whose amino-terminal 600 terminus encoded by the 3' poly(A). The DNA sequence amino acids are similar to RNA14 of S. cerevisiae. Like of intron 4 is highly conserved in Drosophila simulans poly(A}-binding proteins (PABPs), the carboxy-terminal (Langley et al. 1993), and we have detected RNAs similar region of the su(f) protein is proline rich. PABPs also to the 1.3-kb D. melanogaster RNA from the 5' half of have repeated domains that include motifs (RNP-1 and the surf) gene in D. simulans, Drosophila yakuba and RNP-2) found in many RNA-binding proteins (Burd et al. Drosophila mauritiana (data not shown). Furthermore, 1991). These motifs are not present in RNA14 or su(f). In RNA14 is also reported to have RNAs that arise from the yeast, RNAI5 mutants have a similar phenotype to 5' half of the gene (Minvielle-Sebastia et al. 1991}. They RNA14 mutants with effects upon mRNA stability. Un- would also appear to have no encoded stop codon and

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Drosophila modifier gene may give rise to proteins with carboxy-terminal polyl- w that has complex effects on w transcription (Levis et ysine encoded by the 3' poly{A). al. 1984; Zachar et al. 1985; Mount et al. 1988). In w ~ su(f) +, some transcripts are terminated in the 5' LTR of copia, many more are terminated in the 3' LTR, and su(f) Developmental genetics of some go beyond the end of the insertion to the 3' end of The su(f) gene product is required at many different the w transcription unit. Splicing of the latter class of times and places during development, and lack of the transcripts probably produces a small amount of struc- gene product has pleiotropic effects. Temperature-shift turally wild-type RNA that is responsible for pigmenta- studies show that the gene product is required from lar- tion. In w ~ su(f) 1 there is less of the apparently wild-type val stages to eclosion (Dudick et al. 1974; Russell 1974; RNA, indicating that su(f) + activity may stabilize the Jfirgens and Gateff 1979; Wilson 1980). It is also required readthrough transcripts and, therefore, allow processing in the adult female germ line for oogenesis {Perrimon et to occur. al. 1989}. When clones lacking su(f) activity are made in These observations are conflicting, but it is not possi- imaginal discs, there is cell death and disruption of pat- ble to distinguish an effect of su(f) on fl or w ~ transcripts tern determination resulting in duplication of pattern directly from an indirect effect on the expression of an- elements. Flies heterozygous for su(f) ~ and a null allele other gene involved in the expression of fl or w ~. Fur- have short, thin bristles and develop slowly (Schalet thermore, it is not clear whether any RNAs other than 1968). This phenotype is similar to that of the haploin- the apparently wild-type RNAs contribute to eye pig- sufficient Minute mutants, which have mutations of ri- mentation in w ~. With the cloning of su(f), direct anal- bosomal protein genes (Kay and Jacobs-Lorena 1987), and ysis of the activities of the surf) gene products is now that of bobbed mutants, which have partial deletions of possible. rRNA genes (Lindsley and Zimm 1992). This is entirely consistent with a role for su(f) in the synthesis of many different gene products. A role for su(f) in pre-mRNA stability? The similarity between su(f) and RNA14 suggests that su(f) affects the profile of mRNAs from affected alleles su(f) affects RNA stability. A shift to restrictive temper- The most studied examples of phenotypic modification atures in RNA14 temperature-sensitive mutants results by su(f) are the suppression of the bristle phenotype of fl in a rapid decay of actin mRNA levels, as measured in and the enhancement of the eye color phenotype of w ~. RNA blots and a shortening of all poly{A) lengths {Min- However, the effects of su(f) on the stable RNA produced vielle-Sebastia et al. 1991). Although RNA14 appears to from fl and w ~ are contradictory. In fl, a gypsy element affect all yeast mRNAs, the allele specificity of suppres- is inserted in an intron of f and no transcripts can be sion suggests that not all RNAs in D. melanogaster are detected {Parkhurst and Cortes 1985; McLachlan 1986). affected by surf). The effect of surf) on w ~ is also consis- This could be due to an effect on transcription itself or tent with lack of su(f) affecting the stability of some (but on the fate of any transcripts produced. In su(f) mutants, not other)RNAs. apparently wild-type f RNAs are present, and the mutant It is not clear whether RNA14 acts in the nucleus or fl bristle phenotype is suppressed. This suggests that cytoplasm, or both. Lack of su(f) affecting the abundance su(f) + activity results in degradation, rather than pro- of w RNAs of apparently wild-type structure in w ~ but cessing, of fz transcripts containing the gypsy insertion. not affecting the abundance of w § RNAs [data not Parkhurst and Comes (1986) reported that gypsy RNA shown) indicates that su(f) probably acts in the nucleus levels were elevated in mutant su(f) flies and suggested at the level of precursor RNAs, although more indirect that su(f) acted as a negative regulator of gypsy transcrip- effects cannot be excluded. Many genes give rise to more tion. We have been unable to reproduce this result using than one mRNA; and it is possible that by affecting the different stocks of the same mutants, and in a survey of stability of precursor RNAs, su(f) affects the nature of other su(f) mutants, have found no consistent effect the stable RNAs produced. At the very least, this model upon gypsy RNA levels (M. Simonelig and K. O'Hare, in is consistent with the pleiotropic effects seen in su(f) prep.). Dorsett et al. (1989) examined the effect of su(f) on mutants and its requirement at many times and places transcripts from a synthetic gene, where a gypsy element during development. was inserted into an intron downstream from a heat shock promoter. In transfected Drosophila tissue culture cells and in transformed pupae, most transcripts that ini- tiated at the heat shock promoter were terminated at the Materials and methods 5' LTR of the gypsy element. Some readthrough of the Drosophila stocks entire gypsy insertion did occur, and spliced and un- Drosophila stocks were reared on cornmeal/treacle/yeast me- spliced RNAs were detected. In transformed flies the dium at 25~ The collection of PM hybrid dysgenesis-induced abundance of the readthrough RNAs was increased sev- X-linked lethals consists of mutations induced on a fl M strain eralfold in su(f) mutants, which is consistent with su(f) + chromosome using the P strain ~r2 (M. Simmons, pers. comm.} resulting in degradation of the readthrough RNAs. and lethals induced on the X chromosome of ~r2 itself (Simmons In w ~ there is a copia element inserted in an intron of et al. 1984).

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Mitchelson et al.

Genetics puter facility. Motif and pattern searches used the programs described by Henikoff et al. {1990). The revised nomenclature for su(f) alleles from Lindsley and Zimm (1992) has been used throughout: su(f) 8 is su(f) ts6zg, su(f) la is su(f) z6a, and su(f) 26 is su(f) s2. Details of the balancer Acknowledgments chromosomes and most of the marker mutations are given in Lindsley and Zimm (1992). Stocks with PM hybrid dysgenesis- We are particularly grateful to Michael Simmons, who made the induced X-linked lethal mutations were screened using the collection of X-linked lethal mutations. Abe Schalet helped temperature-sensitive lethal allele su(f) 8. Two stocks, 1(1)MS97 with stocks, unpublished data on intra-allelic complementa- and 1(1)MS252, gave l(1)/su(f) 8 offspring at 18~ but not at 29~ tion, and the provenances of su(f) alleles. G.L.G. Miklos, T. The genetic map positions of the lethal mutations were mapped Wilson, E. Gateff, M. Kidwell, and S. Parkhurst also provided by meiotic recombination to the known position of su(f), materials. K. O. was an MRC Senior Fellow; A.M. and C.W. held around position 66. At 25~ neither mutation complemented Student Training Awards from the MRC, and A.M. received lethal surf) mutants but did complement lethal alleles of the further support from the Imperial Chemical Industries Educa- next two distal complementation groups, l(1)sph and 1(1)20 tional Trust. M.S. was a Long-Term EMBO Fellow. This work (Schalet and Lefevre 1976), suggesting that they were mutations was supported by a project grant from the MRC. of su(f) rather than deletions of a region that included su(f). The publication costs of this article were defrayed in part by Females of the genotypes f5 su(f)l/fl 1(I)MS97 and fs su(f)l/fl payment of page charges. This article must therefore be hereby I(1)MS252 showed almost complete suppression of the forked marked "advertisement" in accordance with 18 USC section bristle phenotype, although their bristles were thinner and 1734 solely to indicate this fact. shorter than wild type. This is similar to the "deficiency" phe- notype characteristic of su(f) 1 when heterozygous with a su(f) deficiency (Schalet 1968). By these criteria, both 1(1)MS97 and References 1(I)MS252 behave as lethal alleles of su(f) and have been desig- Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. nated su(f) 27 and su(f) 28. 1990. A basic lor alignment search tool. 1. Mol. Biol. 214: 403-410. Transformation Bankier, A.T. and B.G. Barrel1. 1983. Shotgun DNA sequencing. Techniques in life sciences. Nucleic acid biochemistry (ed. P-element-mediated transformation was as described by Rubin R.A. Flavell), Vol. B5, pp. 1-34. Elsevier Scientific Publishers and Spradling (1982). A 6.4-kb XbaI-BamHI genomic fragment Ltd., Limerick, Ireland. (Fig. 2) was cloned into the vector pW8 (Klemenz et al. 1987) and Brown, N.H. and F.C. Kafatos. 1988. Functional eDNA libraries injected into w ~11s embryos using the plasmid phs~rA2-3wc as from Drosophila embryos. J. Mol. Biol. 203: 425-437. helper. Transformants were identified by their eye color pheno- Burd, C.G., E.L. Matunis, and G. Dreyfuss. 1991. The multiple type, and stocks were established by crossing to balancer RNA-binding domains of the poly(A)-binding protein have strains. One transformant bearing the insertion on the second different RNA-binding activities. Mol. Cell Biol. 11: 3419- chromosome and one with an insertion on the third chromo- 3424. some were obtained. They behaved identically in genetic tests Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehen- as having full su(f) + activity. sive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395. Molecular biology Dorsett, D., G.A. Viglianti, B.J. Rutledge, and M. Meselson. 1989. Alteration of hsp82 gene expression by the gypsy trans- Drosophila genomic DNA was prepared from frozen adult flies poson and suppressor genes in Drosophila melanogaster. as described by Levis et al. (1982). Construction and screening of Genes & Dev. 3: 454-468. genomic libraries, purification of KEMBL4 recombinant clones, Driver, A., S.F. Lacey, T.E. Cullingford, A. Mitchelson, and K. subcloning, and DNA blotting were all performed by standard O'Hare. 1989. Structural analysis of Doc transposable ele- techniques (Sambrook et al. 1989). Preparation of RNA and ments inserted in white and suppressor of forked loci of analysis by RNA blotting using single-stranded DNA probes Drosophila melanogaster. Mol. & Gen. Genet. 220: 49-52. was as described in Levis et al. (1984) and Paterson and O'Hare Dudick, M.E., T.R.F. Wright, and L.-L. Brothers. 1974. The de- (1991). To control for loading, RNA blots were probed with the velopmental genetics of the temperature-sensitive lethal al- ras64B gene (Mozer et al. 1984). DNA sequencing was per- lele of the suppressor-of-forked, l(1)su(f) ts67g, in Drosophila formed by the dideoxy chain-termination method (Bankier and melanogaster. Genetics 76: 487-510. Barrell 1983) using DNA polymerase Klenow fragment (Amer- Fassler, J.S. and F. Winston. 1988. Isolation and analysis of a sham) or Sequenase (U. S. Biochemical). Sequence reactions novel class of suppressor of Ty insertion mutations in Sac- were fractionated on 6% polyacrylamide gels (19:1 mono/ charomyces cerevisiae. Genetics 118: 203- 212. bisacrylamide). Fridell, R.A., A.M. Pret, and L.L. Searles. 1990. A retrotranspo- son 412 insertion within an exon of the Drosophila melano- gaster vermilion gene is spliced from the precursor RNA. Sequence analysis Genes & Dev. 4: 559-565. Gel readings were compiled using the Beckman Microgenie Geyer, P.K., A.J. Chien, V.G. Cortes, and M.M. Green. 1991. package (Queen and Korn 1984). Data bases were searched using Mutations in the su(s) gene affect RNA processing in Droso- BLAST (Altschul et al. 1990), and alignments were made using phila melanogaster. Proc. Natl. Acad. Sci. 88:7116-7120. GAP and BESTFIT programs from the University of Wisconsin Henikoff, S., J.C. Wallace, and J.P. Brown. 1990. Finding protein GCG package (Devereux et al. 1984) on the Medical Research similarities with nucleotide sequence data bases. Methods Council (MRC)-funded Human genome Mapping Project-Re- Enzymol. 183:111-132. source Centre (HGMP-RC) computer facility or the Science and Jenkins, N.A., N.G. Copeland, B.A. Taylor, and B.K. Lee. 1981. Engineering Research Council (SERC)-funded SEQNET com- Dilute (d) coat color mutation of DBA/2J mice is associated

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Drosophila modifier gene

with the site of integration of an ecotropic MuLV genome. Kelly, R.S. Coyne, and V.G. Corces. 1988. The Drosophila Nature 293: 370-374. su(Hw) gene, which controls the phenotypic effect of the Jfirgens, G. and E. Gateff. 1979. Pattern specification in imaginal gypsy transposable element, encodes a putative DNA-bind- discs of Drosophila melanogaster. Wilhelm Roux's Arch. ing protein. Genes & Dev. 2: 1205-1215. 186: 1-25. Paterson, J. and K. O'Hare. 1991. Structure and transcription of Kay, M.A. and M. Jacobs-Lorena. 1987. Developmental genetics the singed locus of Drosophila melanogaster. Genetics of ribosome synthesis in Drosophila. Trends Genet. 3: 347- 129: 1073-1084. 351. Perrimon, N., D. Smouse, and G.L.G. Miklos. 1989. Develop- Klemenz, R., U. Weber, and W.J. Gehring. 1987. The white gene mental genetics of loci at the base of the X chromosome of as a marker in a new P-element vector for gene transfer in Drosophila melanogaster. Genetics 121: 313-331. Drosophila. 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Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability.

A Mitchelson, M Simonelig, C Williams, et al.

Genes Dev. 1993, 7: Access the most recent version at doi:10.1101/gad.7.2.241

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