Download A Drosophila Third Chromosome Minute Locus Encodes

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. S.A.
was supported by the Sven and Lilly Lawski foundation. This work was
supported by a grant from the Swedish Natural Science Research Council
to A.L. and by a grant from the National Institutes of Health to MJ.-L.
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