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Copyright 0 1994 by the Genetics Society of America A Drosophila Third Chromosome Minute Locus Encodes a Ribosomal Protein Stefan Anderson,* Stein SzbaeLarssen,t Andrew Lambertsson,t*'John Merriam: and Marcel0 Jacobs-Lorena§ *Department of Genetics, University of Umed, 9 9 0 1 87 Umed, Sweden, +Department of Biology, Division of General Genetics, University of Oslo, N-0315 Oslo ?, Norway, $Department of Biology, University of California, Los Angeles, California 90024-1606, and §Department of Genetics, School of Medicine, Case Western Reserue University, Cleveland, Ohio 441 06-4955 Manuscript received December 6, 1993 Accepted for publication February 14, 1994 ABSTRACT Minutes ( M ) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3) 95A(Plac92)mutation [allelic toM(?) 95Al. This mutation can be reversed by the mobilization of by insertion of this transposableelement. The the P element, demonstrating that the mutation is caused gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S? genes, and to the Xenopus ribosomal protein S l a gene. At the amino acid level, the overall identity is -78% for all threespecies. This is only the second time that a Minute has been demonstrated toencode a ribosomal protein. M INUTES are a group of over 50 mutations scatteredthroughoutthegenomeand associated with similar dominant visible phenotypes and with recessive lethality. In homozygous or hemizygous condition, Minutemutants are lethal and die at about the time of egg hatching. Flies heterozygous for M i n u t e s are characterized by small bristles and prolonged larval developmental time varying from a few hours to several days delay in extreme M i n u t e s . Other phenotypic characteristics that can be found in extreme M i n u t e s are rough eyes, reduced viability, plexus venation of the wings, fused tergites, deformed or otherwise affected antennae, lowered female fertility, and reduced bodysize (LINDSLEY and ZIMM1992). SCHULTZ (1929) observed in hisclassicalstudy that M i n u t e s are non-additive in their phenotypic effect, i. e., the phenotype of a M I / + ; M 2 / + fly is not more extreme than the phenotypeof any ofthe single mutants. He concluded that the genes code for proteins with similar function (s) . The non-additive property of this type of mutations makes it impossible to determine if a deletion uncovers one or more closely linked M i n u t e s . Bobbed mutations are caused by deletions of rRNA genes and are therefore thought to interfere with the protein synthesizing capacity of Drosophila cells. Based in part on the phenotypic similarity between M i n u t e s and bobbed, RITOSSA et a l . (1966) suggested that M i n utes might affect tRNA genes. Although initially attractive, this hypothesis has since been proven to be incorrect [see SINCWR et a l . (1981) for references]. A much stronger case can be made for the proposal that M i n u t e s ' TOwhom correspondence should be addressed. Genetics 137: 513-520 Uune, 1994) correspondtomutations in ribosomal protein(rprotein) genes [for review, see KAY and JACOBSLORENA 1987). Indirect support of this hypothesis comes from the observation that most cloned ribosomal proteins (rprotein) genes in Drosophila melanogaster have been cytologically mapped to polytene chromosomal regions at or near M i n u t e loci (Table 1). In onecase the M i n u t e to r-protein correspondence has been unambiguously demonstrated when the M(3) 99D mutation was rescued by a cloned r-protein 49 gene (KONGSUWAN et a l . 1985). More recently, QANet a l . (1988) and PATEL andJAcoBsLORENA (1992) found that antisense interference of r-protein gene expression can mimic Minute phenotypes. Based on these experiments they proposed that mutations of the r-protein A1 might yield a Minute phenotype.No direct evidence is available, however, supporting the generality of the correspondence between r-proteinsand M i n u h . A number of Drosophila r-protein genes have been cloned by use of heterologous probes,by use of probes made by enrichment of small mRNAs or by exploring their translational regulation in early embryos (VASLET et al. 1980; FRUSCOLONI et a l . 1983; K A Y and JACOBS LORENA 1985; BROWN et a l . 1988; RAFTI et a l . 1988; MAIU et a l . 1989). One possible approach to unambiguously establish the relationship between M i n u t e s and r-proteins would be to demonstrate rescue of M i n u t e mutants by the clonedgenes, as has been done for rp49. Several attempts have actuallybeen made to rescue M i n utes with cloned r-protein genes but these experiments were unsuccessful (KAY and JACOBS-LORENA 1987; Qm et a l . 1988; K A Y et a l . 1988; DORER et a l . 1991). In this study we take a different approach by cloning a Minute locus without making assumptions about the nature of the 514 S. Anderson et al. TABLE 1 Ribosomal protein genes cloned in D. melanogaster Ribosomal Location protein Reference RP7/8 RPS6 5D 7c5-9 RPS14A&B 7c9 RPSl8 RPA2 RPPlC RP17 15B 21c 21c 29Aa 30DE RPAl RPLl7A RPS15 RPL12 RPSl7 RP2 1 RPs3 RPLl RP49 RPS3l RPS26 RPs19 53CD 58F6-59A3 61F5-62Al 62E 67B1-5 80C 95A 98AB" 99D BURNSet al. (1984) and DENELL (1993); STEWART WATSON et al. (1992); and MACRE (1993) SPENCER et al. (1988); BROWN and LAMBERTSSON (1990) BURNSet al. (1984) OLSON et al. (1993) WIGBOLDUS (1987) MCNABBand ASHBURNER (1993) BARRIOet al. (1993); S. CRAMPTON and F. Lpsu ANDERSON RPs2 NM NM NM * (personal communication) QIAN et al. (1987) NOSELLIand VINCENT (1992) and ASHBURNER (1991) MCNABB BURNSet al. (1984) MAKl et al. (1989) K A Y et al. (1988) WILSON et al. (1993); this report RAFT1 et al. (1988) et nl. (1980) VASI.ET ITOH et al. (1989a) ITOH et al. (1989b) ET A L . (1993) BAUMGARTNER These are the only r-protein loci for which no nearby mapping Minute mutation has been identified. b~~ = not mapped. genes affected. We started with the M(3)95A(Plac92) mutant originally isolated in a P element mutagenesis screen, and cloned the corresponding gene by inverse POlymerase chain reaction (PCR). We found that the phenotype of M(3)95A(Plac92) [allelic toM(3) 95A] is caused by the disruption of a r-protein gene. MATERIALSANDMETHODS Drosophila stocks: The M(3) 95A(Plac92)mutant was recovered from a mutagenesis screen with a stock carrying the enhancer trap P element described in O'KANE and GEHRING (1987). This P element contains the bacterial lac2 and the Drosophila rosy genes. Muta enesis consisted of mobilizing the X-linked P element in a ryFo6 background (kindlyprovided by C. O'KANE) with transposase from the chromosome ryso6Sb P[ry+ A2-3]( 99B)(kindly provided by F. LASKI).Males were crossed to rosy ebony females to select movement of the P element to the autosomes, as described by COOLEY et al. (1988). One Minute rosy' male was obtained from approximately 10,000 progeny and balanced with TMGB. Revertants were obtained by crossing M(3) 95A(Plac92) ry/7y506 Sb P[?' A2-3](99B) males to Df(3R)rya1/MKRS, q Sb or to rosy ebony females,respectively, and the nonStubble rosy progeny were selected and scored for the presence of a Minute' phenotype. The former stocks were kindlyprovided by CAROL MYERS.Other Minute and wild-type stocks were obtained from the European Drosophila Stock Center, UmeP, Sweden. Nucleic acids techniques: For restriction analysis adult Drosophila DNA wasprepared according to the methoddescribed by JOWETI (1986). mRNA for Northern hybridizations and primer extension analysis was isolated directly from crude lysates using magnetic oligo(dT) beads (Dynal A s ;JAKOBSEN et al. 1990).Equal amounts of tissue (25 mg fresh weight) were used in mRNA each isolation. Electrophoresis and nucleic acid blots: Restriction enzyme digested DNAwas electrophoresed through horizontal 0.8% agarose gelsand blotted onto GeneScreenPlus filter membranes (Du Pont-NEN Research Products Inc.). Hybridization and washing conditions were according to the supplier's instructions. Denaturing RNA gels (1.7% agarose, 20 cm long and run at 9OVfor 9 hr), blotting onto GeneScreen filters (DuPont-NEN) and hybridizationswere performed essentially asdescribed by GALAU et al. (1986). Inverse PCR The method used for inverse PCR was basically the one described by OCHMAN et al. (1990). A sample of 0.1 pg DNAwas partially digested with EcoRI (BoehringerMannheim, Germany) for 30 min and heat inactivated for 30 min at 65".The digested DNA wasdiluted 5 times inthe supplied ligation buffer (Boehringer-Mannheim)and ligated at 37" for2 hrwith 1unit ofT4 DNAligase. The ligated DNAwasprecipitated with 2.5 volumes of ethanol and washed in 70% ethanol. The PCR was performed in a 50yl reaction using 2.5 units of Taq Polymerase (US. Biochemical Corp.) in 50mM KCI; 10 mM Tris-HC1 (pH 8.4); 0.1% gelatin; 1.5 mM magnesium acetate; 200 p~ dATP; 200 PM dCTP; 200 PM dTTP and 200 PM dGTP. Two primers were used, 5"ACCACCTTATGTTATTTCATCATG-3' and 5"GACTCCTGGAGCCCGTCAGTATCG 3', from the inverted end repeat of the P element and from the 3' end of the lacZ gene, respectively. These primers were kindly provided by CAROL MYERS. After the PCR reaction the entire sample was run on a 2% low melting agarose gel in TAE (40 mM Tris-acetate, 1 mM EDTA), stained with ethidium bromide and the PCR product was cut out from the gel. Screening a genomicA library: The genomic EMBL3 (Prcmega) A library used was made from an isofemale line of the wild-type stock Shahrinau (LAMBERTSSON et al. 1989). DNA from positive clones was prepared using the small scale lysis method described by MANIATIS et al. (1982). Screening a cDNA library: The cDNA library used was a A gtlO library with cDNA inserts from RNA prepared from head tissue from adult wild-typeflies; the library was made by CHARLES ZUKER,University ofCalifornia, San Diego, and kindly provided byD. LARHAMMAR, BMC, Uppsala. In situ hybridization: In s i t u hybridizations to salivary gland polytene chromosomes were essentially asdescribed by PARDUE (1986). Probes: All probes for Southernblot hybridization were labeled with[3'P]dCTP by primer extension using Promega Prime-a-GeneSystem (Promega) to a specific activity of 1-2 X loHcpm/pg. The probes for in s i t u hybridization were labeled with t3H]dCTP in the same way. For Northern blot hybridizations double stranded probes were labeled as described above. In addition, strand specific probes were generated using biotinylated single-stranded templates (sense and antisense) bound to magnetic streptavidincoated beads (Dynabeads "280 Streptavidin, Dynal M) in a standard random priming reaction (ESPELUND et al. 1990). Sequencing: Genomic DNAand cDNAwere subcloned into pUC19 and then sequenced by the chain terminationtechnique (SANGER et al. 1977) usingthe Taq Track SequencingSystem (Prcmega) and [35S]dATP following the instructions of the supplier. After reamplification and MagicPCR (Promega) minipreparation, PCR products were cloned into the pGEM-T vector (Promega) and sequenced by the femtomolesequencing system (Promega). Acrylamide gels (5%) of 38 X 50 cm were used. Primer extension analysis: The primer extension was carried out on mRNA isolated from late third instar larvae using Drosophila M i n u l r s and r-Protcins .?I5 avian myeloblastosis virus (AMV) reverse transcriptase (Promega) and an end-labeled rpS3-specific oligo (.5'-CTTGCr GTITCTTGGAA-3') complementary to the last 16 nucleotides of the rpS3 first exon. Following annealing to primer in 40msc KCI, 50 mu Tris, pH 8.3, at 42", reactions were adjusted to 50 mu Tris, pH 8.3,40 mu KCI, 7 ms,f MgCI,, 1 msl dithiothreitol, 0.1 mg/ml bovine serum albumin, 1 mxl dNTP, 50 pg/ml actinomycin D, 0.25 unit/\ll RNasin, 0.5 unit/pl AMV reverse transcriptase, and incubatedfor 2 hr at 42".The reaction products were precipitated with ethanol and redissolved in formamide loading buffer. The complementary sequence was produced using the ~1S3-specific oligo described ahove and a PCRamplified genomic sequence as template. Dideoxy sequencing reactions were performed on biotinylated single-stranded templates hound to streptavidincoated magnetic microspheres (Hl!I.T\l.I?\N et 01. 1989). Primer extension producg were electrophoresed on 8% polyacrylamide gels. Nucleotidesequence accessionnumber: The accession number for therjlS3 gene andits flanking regions is X72921. RESULTS Localizationandidentification of the P elementinduced Minute mutation: The cytological position of the P element-induced Minute mutation was determined by in situhybridization to salivarygland polytene chromosomes. These in situ hybridizations showed that there isonly one IacZcontaining P element in the M ( 3 ) 9 5 A ( P l a c 9 2 ) r y / T M b R , T h stock and that the element has inserted in region 94F/95A ofthe third chromosome (Figure 1A). A 5.6kb RamHI fragment containing the gene disrupted by the P element insertion (see below) was also used for i n s i t u hybridization to salivary gland polytene chromosomes and the hybridization was localized to region 94F/95A on a wild-type chromosome (Figure 1B), the same region of hybridization as the lacZ probe. Both the rosy' insert and the FI(x'w I .-(:hrotnosorn;tl localization of the P element inMinute phenotype were mapped by recombination , chromosomes sertion. (A) i\1(3) 95'4 (Plnc92) ~ / T M f i B 7% (data not shown) to map position 111-73, approximately hybridized with a probe that contains the Eschrrirhin coli InrZ 2 map units the to right of ebony, which is in good agreesequence. The IncZ sequence hybridizes to region 94F/I)51\ only on the M ( 3 ) Y5A(PlncY2) ?ychromosome. (R) Wild-hpe ment with the cytogenetic localization. chromosomes hybridized with the .5.fi-kb BnrnHI fiagment (1-1 To determine the relationships among the M(3)Figure 4A). Hybridization is detected at 94F/I).5rZ. 95A(Plac92) mutation and other Minutes in the region, P The P element insertion causes the Minute phenoelement-induced M(3)95A(Plac92)?y/TM6R,Th females type: To determine whether the Minute phenotype in were crossed to males of two other Minute stocks in our the M ( 3 ) 9 5 A ( P l n c 9 2 mutant ) is caused by a P element collection: I(3)ac es M(3)95Al/TM6R, Th and M(3)95A2/ TMbR, Th. We never found any non-Tubby Minutes in the insert, a dysgenic cross was set up to mobilize the Pelprogeny of these crosses. Since Minutes are non-additive ement and thereby revert the phenotype to wild type. recessive lethals, the noncomplementation in these M ( 3 ) 9 5 A ( P l a c 9 2 ) q / T M b B , 7'1) females were crossed to 7 y 5 ' " S h P[,' A 2 - 3 ] ( 9 9 R ) / T M 6 R , Uhx males, and crossesshows that M(3)95A(Plac92),M(3)95AI and M ( 3 ) 9 5 A ( P l a c 9 2 ) q1/7y5'"'S b P[?+ A 2 - 3 ] ( 9 9 B ) males M(3) 95A2are all allelic mutations. M ( 3 ) 9 5 was A named M(3)zu prior to 1989 (ASHBURNER were collected from the progeny. These maleswere crossed to Df(3R)q1*'/MKRS, 9 S h females, and non1989). M(3)70 was localized cytologically to 95A1by BRODERICK and ROBERTS(1982) and to 94D-E by VAssrx Stubble rosy males and females were selected and scored et al. (1985). Both groups used duplications for this refor Minute or wild-type phenotype. Revertants displavgion and since 94F is a small band, it might be difflcult ing a Minute+ phenotypewere found, thusshowing that to distinguish if the break point is close to or in band the Minute phenotype can be reverted by mobilization 94F. Unfortunately, the morphology of our i n sifzL hyof the Pelement.Revertant stocks wereestablished from bridizations does not allow us to distinguish between single males. DNA from one such revertant line is anahybridization to 94F or 95A. lyzed in Figure 2, lane 3. All revertants from this cross S. Andersson rt nl. 516 1 2 3 4 kb m HS Rr 19.9 X1 m 5.6 FIGURE 2.-Southern blot analysis. DNA from (1) a wild-type stock, (2) the original M ( 3 ) 95A(Plnr92) ?r/TMfiR, Tb stock, (3) a revertant stock and (4) the non-Stubble Minute rosy stock was digested with RnmHI. The blot was probed with a 5.6-kh RnmHl fragment containingsequences surrounding the point of Pelement insertion ( r-f. Figure 4A).The last two strains were recovered from a dysgenic cross designed to mobilize the inserted Pelement (see first paragraph of the RESL!I,TSsection). were later accidentally lost. Additional revertants were o b tained from another cross. Fifteen males of the genotype M(3)95A(Plac92) 9/lySffi Sb P[ry+ A2-3](99B) crossed with rosy ebony females yielded 135 exceptional progeny simultaneously rosy and Minute+.Since premeiotic P loss results in clusters of identical revertant progeny, only one revertant was retained from each father for further study. One of these, revertant #4, was used for primerextension and Northern analyses (see Figures 4 and 5 ) . Identification of sequences disruptedby insertion of the Pelement: Cloning the DNA surrounding the point of P element insertion was initiated through obtaining the genomic sequence flanking the 5' side of the insert by inverse PCR. DNAfrom M(3) 95A (Plac92) ry/TMbB, T b flies was partially digested with EcoRI, religated, and PCR amplified yielding a =700-bp product. To determine if this product originated from DNA adjacent to the P element insertion, genomic Southern blots of DNA from M(3) 95A (Plac92)ry/TMGR, TI, and wildtype flies were hybridizedwith the 700-bp PCR product. The autoradiogram showed an additional band in the lane with DNA from M(3)95A(Plac92) ry/TMGR, TI) not detected in the lanewith wild type DNA (data not shown). This additional band also hybridized to a lac2 probe and,as expected, this probe gave n o hybridization to wild type DNA (data not shown). These results indicated that the 700-bp PCR product contained a Drosophila DNA fragment acfjacent to the inserted P element in the M(3) 95A(Plac92)mutant. The PCR product was used to screen a Drosophila genomic library. Several of the positive A clones had a 5.6kb RamHI fragment in common, which hybridized to the PCR product. A band of approximately the same size was also seen when thePCR product was hybridized to RamHIdigested genomic wild-type DNA (not shown). The 5.6-kb RamHI fragment was used to probe Southern blot9 containing RamHI-digested DNA from a wild type strain, from theoriginal Minute strain, from a revertant strain and from the non-Stubble Minute rosy strain (Figure 2). The last two strains were recovered from the first dysgenic cross described above. The wildtype strain and several of the revertantstrains show only HS Rr X1 1 1 1 1 MNAN~~ISKKRKWSWIFKAELN~FLTRELAEDGYSOVEVRVTPFGTEI M-A-VQISKKRKFVADGIFKAELNEFLTRELAEDGYSGVEVRVTPTRTEI M-A-VQISKKRKWADGIFKAELNEFLTRELAEDGYSGVEVRVTPTRTEI M-A-VQISKKRKFVADGIFRAELNEFLTRELAEDGYSGVEWWTPTRTEI 51 IIR~TRTQQVL~KGRRIRELT&QKRFNFETGR!~!ELYAEKVAARGLCA 49 IILATRTQNVLGEKGRRIRELTAWQKRFGFPEGSVELYAEKVATRGLCA 49 IILATRTQNVLGEKGRRIRELTAWQKRFGFPEGSVELYAEKVATRGLCA 49 IILATRTQNVLGEKGRRIRELTAWQKRFGFPEGSVELYAEKVATRGLCA 101 IAQAESLRYKLTGBLAVRRACYGVLRYIMESGMGCEVVVSGKLRGQRAK 99 IAQAE~LRYKLLGGLAVRRACYGVLRFIMESGMGCEVVVSGKLRGQRAK Rr 99 IAQAESLRYKLLGGLAVRRACYGVLRFIMESGAKGCEVVVSGKLRGQRAK X 1 99 IAQAESLRYKLLGGLAVRRACYGVLRFIMESGAKGCEVVVSGKLRGQ~K m HE m 151 SMKFVDGLMIHSGDPCNTATFUWLLRQGVLGIRVK~LP~DPKNKI H S 149 SMRFVDGLMIHSGDPVTAVRIWLLRQGVLGIICVKIMLPWDPTGKI Rr 149 SMRFVDGLMIHSGDPVNYYVDTAVRHVLLRQGVLGIKVICIMLPWDPSGKI X1 149 SMKFVDGLMIHSGDPVNYWDTAVRHVLLRQGVLGIICVKIMLPWDPSGKI m 201 GPKKPLPDE~~SVVEPKBEK~YETPETEYKIPPPSKP-LDDESE~VL HS 199 GPKRPLPDHVSIVEPKDEILPTTPISEQK~~KPE---LPAMPQPVPTA Rr 199 GPKKPLPDHVSIVEPKDEILPTTPISEQKGGKPE---PPAMPQPVPTA X 1 199 GPKKPLPDHVSIVEPKDEIVPTTPISEQKAAKPEQPQPPAMPQPVATA FIGURE S.-RPSS amino acid alignment. Alignment of the predicted RPS3 amino acid sequences from D.mdmogmlpr (Dm), Ifoma snt/ipns (Hs) ,Rntlrr.7 mtt?c.s (Rr) and Xpn@:.s / m v i . s (XI). Light gray shaded regions represent sequence identitywhile the darker shadedregionsrepresent consenative amino acid changes. Amino acid substitutions that do not fit the consensus are not shaded and gaps are indicated by dashes. one hybridizing 5.6-kb fragment (Figure 2, lanes 1 and 3, and data not shown). The M(3) 95A(Plnc92) and the non-Stubble Minute rosy strains yield two fragments: one 5.6 kb (originating from the balancer chromosome) and one larger(Figure 2, lanes2 and 4). For both Minute strains the larger fragment hybridizes to a lacZ probe (not shown), indicating that theextra band was caused by insertion of the P element (14.7 kb). The second fragment in the non-Stubble Minute rosy strain is -4 kb smaller thanthe 19.9-kb band in the M(3) 95A(Plac92)DNA (Figure 2, lanes 2 and 4), suggesting that in the former strain part of the P element construct was deleted. Part of the 9' gene must have been deleted in this strain, since the flies are rosy; this was not further analyzed, however. Sequence and primer extension analyses: To determine the identity of the geneaffected by the Pelement mutation approximately 60,000 phages froma Drosophila cDNA library were screened with the 5.6-kb RamHI fragment as a probe. Approximately 60 positive clones were detected, of which 17 were rescreened and further examined. Two of these cDNA clones were sequenced and found to be identical, except for the length of the poly(A) tail. The deduced amino acid sequence from the Drosophila cDNAs was73 aminoacids shorter at the 5' end than similar cDNAs from other organisms, suggesting that the sequenced cDNAs were incomplete (see below). Therefore, genomic DNA flanking the P element insertion point was sequenced. This sequence suggested an open reading frameof 12 codons, followed by a putative intron (see below) and an additional open reading frame overlapping with the partial cDNA (results not shown; accession no. X72921). In all, 1664 Drosophila MinwtPs and r-Proteins .5l7 .. Revertant M(3)95A(PIac92) Wild type A C G T 1 i .- f FIGURE 4.4rganization of the Drosophila rpS3 gene. (A) Restriction mapof 5.4 kb ofa A clone harboring the rj,S3 gene. The P element (not shown to scale) insertion siteis indicated. The proteincoding regions of the $73 gene are shown as black boxes, stippled boxes indicate noncoding parts of the exons, and the thin line in between the boxes represents the intron. The 5.CFkb RnmHI-RnmHI fragment mentioned in the text extends from the RnmHI site 500 bp tlpstream of the transcription start site to a RnmHI site 5.6 kb further downstream not shown on the map. (R) Primerextension analysis. mRNA from Oregon-R, M(3)95A(Phc92)ly/TMhR, Tband homozygous revertant#4 late third instarlana was extended from the primer describedin M K I ' E R ~ ~ ~ S AND LWTI-IODS.The major and minor primer extension products are at positions -36 and -40 from the translation initiation codon, respectively. The filled arrowhead shows the position ofthe Pelement insertion between nucleotides -20 and - 19.The first open reading frame (OW) is indicated by a rightward arrow. The translation start codon in the first ORF is also indicated. adenvlation sequences AATAAA separated by 14 nuclenucleotides (including the intron)of genomic DNA were otides are found at the end of the cDNA, starting at sequenced. No polymorphisms were found between the positions +839 and +859. In the Xenopus sequence two cDNA and the overlapping genomic DNA sequences. AATAAA sequences are found separatedby a single A. The sequence from the cDNA clones was compared Based o n these comparisons we conclude that theDrowith the sequences in the EMBL Data Library. The Drosophila sequence is highly similar to r-protein genes sophila cDNA encodes a r-protein homologous to the from other organisms, the best scores being obtained for mammalian RPS3 and Xenopus RPSla. Figure 4A summarizes the organization of the Drohuman rpS3 (ZHANCet al. 1990), rat rpS3 (CHANet nl. sophila rj1.53 gene, its flanking regionsand indicates the 1990) andXenopus rpSIa (Dr CRISTISA et nl. 1991; insertion siteof the Pelement. There are P. PIERANDREI-AMAIDI, unpublished). The human and two open readthe rat proteins are 243 amino acids long, while the Xeing frames, 36 and 702 nucleotides long, respectively, nopus and the Drosophila proteins are 246 amino acidsseparated by a single 256-nucleotide long intron. The existence and extent of the intron, which contains an long (Figure 3). The amino acid sequence of the Droin-frame stop codon, is based in part on interspecific sophila r-protein is strikingly conserved whencompared comparisons. M'n.sos et nl. (1993) have recently seto those of human, rat and Xenopus; overall the identity quenced a full length rf1.53 cDNA from D. melnnognster is -78% for all three species. In particular, the region in which the intron is spliced out at exactlv the place fromaminoacid 7-218 in D. melnnognster (amino predictedfrom oursequence analysis. Furthermore, acid 5-216 in the vertebrate sequences) differs only in both the splice donor and and acceptor sequences and 26/212 amino acids (-88% identity). The 28 amino the branchpoint tetranucleotide match acids at the end of the deduced protein sequence are very well the Drosophilaconsensussplicesequencesdescribed by highly divergent and differ bothin charge and polarity. M o c s ~et al. (1992) (results not shown; accession no. Similarly, thenucleotidesequence in the AT-rich X72921). The Drosophila +73readingfmme is terminated S'-untranslated region of the cDNA is not particularlv bv hvo stop codons in the sameframe, separated by three conserved inthe fourspecies, although somesimilarities nucleotides. The deducedprotein is 246 amino acids long can be found (results not shown). Two canonical poly- and has a predicted molecular mass of 27.5 kD. I n comparison, the calculated molecular mass of the human and rat RPS3 is 26.7 kD, and that of Xenopus is 27.0 kD. By sequencing the PCR product and comparing the sequence with the genomic sequence, the insertion of the P element was found to be between the G and T at position -20 and -19, respectively (Figure 4R). Thus, the insertion is in the transcribed portion of the rj)S3 gene behveen the transcriptionstartsite(s) andthe translationinitiation codon(Figure 4B). Northern analysis (see below) suggests that P element insertion severely depresses transcription of rj1.53. The results from the primer extension analysis are shown in Figure 4B. There are two transcription start sites at positions -36 and -40, position -36 being the one most frequently used. The start sites are in a pyrimidine rich tract, which is typical for mammalian and several Drosophila r-proteingene capsites (STEWART and DENELL 1993; MAGER1988). The presence of a polypvrimidine tract has been found to be important for both translational regulation and promoter function(CHLINC; and PERRY 1989; MOURA-NETOrt nl. 1989; HARII-IXRAN and PERRY 1990; L ~ w e nl. t 1991). However, not all Drosophila r-proteins have this motif. There are no apparent TATA o r CAAT motifs typically found in promoter regions of genes transcribed by RNA polymerase 11. The lack of a TATA box is not unusual for r-protein genes. Figure 4B also reveals thatthe levels of the two rj1S3 primer extension products are reduced in M(3)95A(Plnc92) in comparison with wild type. The lack of an internal quantitation control does not allow for a quantitative assessment of the extentof the reduction in this experiment. However, the apparent restoration of the levels of primerextension products in the revertant (Figure 4B) and the reduction of transcript levels in M(3) 95A(Plnc92)detected on Northern blots (see below) are all consistent with the notion that the phenotype of M(3) 95A (Plnc92) is caused by the disrup tion of the rj~S3gene by the inserted P element. Northern analysis: Figure 5 shows the analysis of late third instar larvae poIy(A)' RNA from wild type, M(3) 95A(Plnc92) q1/TM6R,TI,and revertant #4. The blot was hybridizedwith a single-stranded rpS3 probe and with a Drosophila a-tubulin probe as loading control. The results indicate that the amountof both rpS3 transcripts in mutant larvae is reduced whereas rpS3 mRNA abundance in revertant larvae is indistinguishable from wild type. Therefore, the insertion of the P element result$ in a reduction of the rpS3 mRNA abundance. The high resolution of our Northern blots allowed the identification of two rpS3 transcripts in a p proximately equalamounts, differingin sizeby about 40 nucleotides. The proportionof the two transcripts does not change during development (results not shown). Whether thetwo transcripts are generated by alternative splicing or by other means is presently being investi- 940 900 rpS3 a-tub FIGURE 5.-Northern blot analysis. Late third instarlarval poly(A)' RNA from Oregon-R, " 3 ) 95A(Plnc92) ry/TMhB, 7% and homozvgorls revertant # 4 was separated on a 1.7% agarose gel at 4.5 V/cm. Polv(A)' RNA equivalent t o 5. mg fresh tissuewight was loaded in each lane. After elcctrophorcsis the RNA was blotted onto a CeneScrccnPlus filter and probed with a sense specific rj,s3 cDNA probe. After removing the rpS3 probe, the blot was hybridized with a 11. mnhnngm/Pr cy-tubulin probe to verify the amounts of RNA loaded i n C;Ich lane. gated and will be reported elsewhere. N o other transcripts were observed on the Northern blots described above when probed with the sense or antisense r j ~ S 3 cDNA, with the double-stranded upstream 2.4-kb Hind111 fragment or with the 1.85-kb BgnI fragment downstream of r j ~ S 3( $ Figure 4A; data not shown). This suggests that no other transcripts are encoded by the region surrounding rj~S3. We conclude that M(3)95A(Plnc92) is a mutation in the r j ~ 5 3gene. DISCUSSION Minutes and ribosomal proteins: So far most Drosophila r-protein genes have been mapped to regions near Minutr loci (Table 1). Our results are consistent with the hypothesis that MinutPs aremutations in r-protein genes. The fact that the M(3)95A(Plnc92) is caused by the insertion of a P element within the transcription unit of r j ~ S 3the , reversibility of the phenotype upon mobilization of the P element, the respective decrease and restoration of rj1.73mRNA abundance inmutant and revertant flies, and the high similarity of the gene disruptedby the Pelementto vertebrate r-proteins argue very stronglythatdisruption of Drosophila r-protein gene S3 results in a Minute phenotype. Thisis only the second example of assignment of a Minute mutation to disruption o f a r-protein (the first example was by KONGSUM'AN rt nl. 1985). These two examples, do, however, not prove the generality of the relationship. It is possible that not every mutation in a ribosomal protein results in a Minute phenotype. For instance, rpL1 (R\m rt nl. 1988) and $1 7 (M(:NABRand ASHRURNER 1993), map to regions where a Minute has not been identified. However, this does not rule out the possibility that mutations in rjjL1 or r p l 7 yield Minute phenotypes. It is possible that the mutant has not yet Drosophila r-Proteins Minutes and been found or that theexpression of r p L l and r p l 7 is not haplo-insufficient, or that the gene is haplo-lethal. If the genes arenot haplo-insufficient, recessive (rather than dominant) Minute mutations could be found. If thegenesare haplo-lethal, mutationsthatreduce (rather than eliminate) gene function might lead to Minute phenotypes. Several attempts have been made to demonstrate that clonedr-protein genes correspond to Minute mutations mapping near where to they had been cytologically localized. This was successful only in the case of M(3)99D, which was rescued by rp49, but not successful in a numberof other cases (see Introduction). The reason for the latter unsuccessful rescue attempts might have been that the Minutes in question were deletions uncovering two or more r-protein genes. The present approach of directly cloning Minute mutations circumvents this type difficulty. It is possible thatmutations in genes other than r-proteins lead to Minute phenotype. VOELKER et al. (1989) found that M ( 1 ) 1 Bencodes a 3.5-kb message that could encode a -110,000-kD protein. This is far larger than anyknown Drosophila r-protein, which range in size from 11,000 to -50,000 kD ( CHOOIet al. 1980). Therefore, it seems unlikely that the M ( 1 ) l B phenotype is caused by a mutation in a r-protein gene. Partial inactivation ofgenes involved in protein synthesis such as aminoacyl-tRNAsynthetases or protein synthesis factors might yield a Minute phenotype. Alternatively, any mutations that affect ribosome assembly and transport, aswell as the structural makeup of ribosomes, might result in a phenotype similar to Minutes. The bobbed (ribosomal RNA genes) and the mini (5s RNA genes; KAY and JACOBS-LORENA 1987) loci are two examples. In the future, the cloning andcharacterization of other Minutes might further clarify these issues. Are ribosomal proteins multifunctional?: Interesting recent results indicate that mutationsin r-protein genes may lead to uncharacteristic phenotypes suggesting that ribosomal proteins are not simply required structural elements butalso are implicated in regulatory processes that may be importantin normal development. Thus, in Drosophila, rpS6 behaves as a tumor suppressor gene (TSG) and is encoded by the aberrant immune response 8 ( a i r 8 ) locus (WATSON et al. 1992; STEWART and DENELL 1993). The RpS3 protein have been found to have AF' (apurinic andapyrimidine) endonuclease activity (WILSON et al. 1993). Overexpression of the Drosophila r-protein S15a suppresses a mutation in the Saccharomyces cerevisiae cdc33 gene, which encodes the capbinding subunit of eukaryotic initiation factor 4F (eIF4F LAVOIE and LASKO 1993). Mutations of cdc33 lead to arrest in the cell cycle at the G, to S transition. string of pearls (sop) is a recessive female sterile mutation in D . melanogaster that arrests oogenesis at stage 6. The gene product is identified as the Drosophila homolog to RpS2 of yeast and rat, the equivalent of pro- - 519 karyotic r-protein S5 (CRAMPTON and LASKI1993). Finally, rpL19 is overexpressed in human breast tumors and this overexpression is independent of other ribosomal proteins (HENRY et al. 1993). Additional ribosomal proteins have been implicated in regulatory processes that may be important in carcinogenesis [see HENRY et al. (1993) for references].Since the exact functions of the ribosomal proteins mentioned above is not known, the link between phenotype and gene function remains to be determined. Conclusions: The evidence presentedhere shows that M(3)95A(Plac92) is a mutation in the r-protein gene S3. M(3) 95A(Plac92) is only the second Minute to be characterized molecularly and shown to encode a r-protein. Many more Minute. mutations needto be analyzed to determine the generality of this correlation and to determine whether mutations in non-ribosomal genes can lead to the Minute phenotype. The present report demonstrates that analysis of P element insertional mutants is a fruitful approach to address these questions. Authors S.A. and S.S.-L.have contributed equally to this work. We thank THOREJOHANSSON for expert technical assistance and KARIN EKSTROM for making the in situ hybridizations and providing fly stocks and ANDEM BLOMQVIST for screening the cDNA library, and KARIN BLOCK for interpreting the in situ hybridizations. We are grateful to CAROL MYERS for providing primers andfly stocks, and forfruitful and stimulating discussions. We thank the editor (ROB DENELL) and two anonymous reviewersfor many useful suggestionson the manuscript. 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