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Copyright 1999 by the Genetics Society of America
Molecular Cloning and Tissue-Specific Expression of the mutator2
Gene (mu2)inDrosophila melanogaster
Armin Kasravi,* Marika F. Walter,* Stephanie Brand,
†
James M. Mason
‡
and Harald Biessmann*
*Developmental Biology Center, University of California, Irvine, California 92697,
‡
Department of Anatomy and Physiology,
University of Dundee, Dundee DD1 4HN, Scotland and
†
Laboratory of Molecular Genetics, National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Manuscript received November 25, 1998
Accepted for publication April 9, 1999
ABSTRACT
We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila
melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis
during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating
somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2
represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2
does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing
agents.Wehavemappedandclonedthemu2geneandrescuedthemu2phenotypebygerm-linetransforma-
tion with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2
encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear
localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the
gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the
nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also
prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are
consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2
protein may be involved in controlling chromatin structure and thus may influence the processing of
DNA DSBs.
T
HE integrity of the genome is of great importance ferently in females. A gene, mutator2 (mu2), has been
to cycling cells. Treatments that result in DNA dou-
describedinD.melanogaster,whichaffectstheprocessing
ble strand breaks (DSBs) trigger a nuclear signaling
of DSBs in the female germ line (Mason et al. 1984).
pathway to the cell cycle machinery that causes cell
Genetic analysis has identified several genes in this or-
cycle arrest. While mitotic cell cycle checkpoints are
ganism that play a role in meiotic recombination and
beginning to be understood in a variety of organisms
are also required for DNA repair (Baker et al. 1976b;
(Carr 1996; Elledge 1996; Paulovich et al. 1997),
Hawley et al. 1993; Ferguson et al. 1996). mu2 is not
much less is known about meiotic cell cycle control.
allelic to any of these genes and represents a novel
Irradiation of postmeiotic germ cells has been widely
component in the processing of DSBs in meiosis.
used to study the fate of broken chromosomes in Dro-
mu2 may be involved in maintaining chromosomal
sophila melanogaster (Muller 1938, 1940; Muller and
integrity in the female germ line. The original mu2
a
Herskowitz 1954; Roberts 1975). These experiments
allele was found in a laboratory stock because its pres-
led to the definition of telomeres on the basis of their
encesignificantlyincreasedtherecoveryofspontaneous
function in capping chromosome ends. A broken chro-
yellow mutations (Mason et al. 1984). The yellow mutant
mosome without a telomere on one end could not be
frequencyinducedby irradiationofmatureoocytes with
recovered unless it had acquired a new telomere by
5Gyofg-rays increases z2-fold in heterozygotes and
fusion with another chromosome fragment. For techni-
20-fold in homozygotes compared to wild type. Unlike
cal reasons female germ cells have been used much less
the mutants recovered from thewild-type control, these
extensively in mutagenesis experiments (Parker and
radiation-induced mutants from mu2
a
females resulted
Williamson 1976).
fromterminal, i.e.,one-break, deficienciesthat havelost
Whileterminally deletedchromosomes havenotbeen
a tip of the original chromosome and have not been
recovered amongoffspring ofirradiated males, itis now
“capped” via rearrangement (Mason et al. 1984; Biess-
apparent that broken chromosomes are processed dif-
mannetal.1990).Thebrokenchromosomeendsrecede
at a rate of z75 bp per sexual generation, probably due
to the inability of the DNA replication machinery to
Corresponding author: Harald Biessmann, Developmental Biology
completely replicate the ends of a linear DNA molecule
Center, University of California, Irvine, CA 92697.
E-mail: hbiessma@uci.edu
(Biessmann and Mason 1988; Biessmann et al. 1992).
Genetics 152: 1025–1035 (July 1999)
1026 A. Kasravi et al.
pfu/plate and screened sequentially with subcloned BamHI
While mu2
a
increases the half-life of a radiation-
genomic fragments from the yeast artificial chromosome YAC
induced lesion in oocyte chromosomes from 10–20 min
N77-23 (see Figure 1). Hybridization was done as described
to 20 hr and potentiates the recovery of terminal defi-
(Walter et al. 1995). DNA fragments to be used as hybridiza-
ciencies from irradiated females, it has no discernible
tion probes were purified by gel electrophoresis in low-melt
agarose, and 20–50 ng were used for labeling by random
effect in the male germ line (Mason et al. 1997). Chro-
priming reaction (Prime-it II; Stratagene). cDNA inserts were
mosome breaks in irradiated sperm are repaired nor-
subcloned from purified phage DNA into pBluescript (Strata-
mally evenafter fertilizingmu2 eggs,suggesting thatthe
gene) for further analyses.
primary defect in the mu2 mutant is not in DNA repair
Rapid amplification ofcDNAends: RACE was performedby
per se. However, when mu2 oocytes are irradiated, radia-
PCR with two different cDNA libraries: the 0–8 hr embryonic
cDNA library (Nicholas Brown) and the ovarian cDNA library
tion-induced lesions in the maternal chromosomes re-
in lgt22A (Stroumbakis et al. 1994). As primer combination,
main unrepaired for long periods of time (Mason et al.
we used the primer mu2cDNA-10 (59 CGAATCCGCYACTG
1997).
TCGTGG 39) located 339 bp downstream of the 59 end of the
We describe here the somatic phenotype of mu2
a
and
longest mu2 cDNA (26H9; GenBank accession no. AF108206)
show that mutant females exhibit a significant increase
and a corresponding inward-facing primer from either the
NB40 cloning vector or from the lgt22A phage, depending
in somatic recombination. We also report the cloning
on the library to be screened. PCR fragments were digested
and molecular analysis of the mu2 gene as well as its
with desired restriction enzymes, cloned in pBluescript, and
temporaland spatialexpression pattern.Our molecular
sequenced.
results are consistent with the observed phenotype of
RNA isolation and Northern blots: Total RNA was purified
mu2
a
in the germ line and somatic tissues and provide
from various developmental stages with the TRIzol reagent of
GIBCO/BRL (Gaithersburg,MD). Poly(A)
1
RNA wasaffinity-
further insights into potential functions of the encoded
purified with the polyATtract mRNA isolation system (Pro-
polypeptide.The presentresultssupport themodel that
mega, Madison, WI) and electrophoresed at 2 mg/lane on
mu2 is a structural component of the oocyte nucleus,
1% agarose, 0.66 m formaldehyde gels. RNA was transferred
whereit maybeinvolved incontrollingchromatin struc-
for 36 hr by capillary action in 203 SSC (3 m NaCl, 1.5 m
ture and thus may influence the processing of DSBs.
sodium citrate) to Hybond N
1
nylon sheets (Amersham, Ar-
lington Heights, IL), crosslinked by UV (Stratalinker;
Stratagene), and hybridized to a random-primed mu2 cDNA
probe as above.
MATERIALS AND METHODS
RNA in situ tissue hybridizations: mu2 expression was stud-
ied by whole-mount in situ hybridization using a digoxigenin-
Genomic DNA isolation, Southern blotting, and hybridiza-
labeled antisense RNA probe (Tautz and Pfeifle 1989).
tions: Genomic DNA from wild-type and various deficiency
Sense or antisense digoxigenin-labeled RNA transcripts were
strains was isolated and used for Southern blotting and for
generated from the mu2 cDNA-26H9 in pBluescript using
establishing genomic DNA libraries as described previously
(Walter et al. 1995). the digoxigenin RNA labeling mix (Boehringer Mannheim,
Indianapolis). The sense strand (negative control) was pre-Recombinant phage libraries: Recombinant phage libraries
were generated by partially digesting genomic DNA with pared by adding 2.5 ml water, 2 ml53 T3 buffer, 1 ml103
digoxigenin NTP, 0.5 ml 100 mm DTT, 1 ml RNasin, 1 mg NotI-Sau3A, partially filling the Sau3A sites with Klenow enzyme
and dGTP and dATP, and ligating 1.2 mg of DNA with 1.2 linearized cDNA, and 1 ml T3 polymerase (Promega). For the
antisense probe, HindIII-opened cDNA and T7 polymerasemgoflBlueSTAR phage arms with partially filled XhoI sites
(Novagen, Madison, WI). Recombinant phage DNA was pack- (Promega) were used. The reactions were incubated for 2 hr
at 378. Hybridization to formaldehyde-fixed tissues was doneaged with Gigapack II Plus (Stratagene, La Jolla, CA) and
plated on Escherichia coli DB1316 (Wertman et al. 1986) at in 50% formamide, 53 SSC, 100 mg/ml salmon sperm DNA,
50 mg/ml heparin, and 0.1% Tween 20 for 3 days at 558. After5 3 10
4
plaques per plate.
DNA sequencing and sequence analysis: Sequencing was extensive washing in hybridization solution (minus salmon
sperm DNA) for 2 days with six changes, with a 1:1 hybridiza-done from double-stranded Bluescript vectors by the dideoxy-
nucleotide termination technique, using Sequenase 2.0 ac- tion solution/PBT (PBS and 0.1% Tween 20) for 20 min, and
fivewashesinPBTfor25mineach,thesampleswereincubatedcording to the manual of United States Biochemical Co. Se-
quence analyses were done with the DNA Strider program overnight at 48 in polyclonal sheep anti-digoxigenin Fab frag-
mentsconjugated toalkalinephosphatase(Boehringer Mann-(Marck 1988). Oligonucleotide primers were synthesized by
Genosys International (The Woodlands, TX). GenBank heim)diluted1:2000inPBT.Specimenswerewashedsixtimes
in PBT for 30 min each. Prior to detection, the samples weresearches were done with BLAST (Altschul et al. 1990), analy-
sis of the protein sequence was done with PROSITE (expasy. washed twice in an alkaline solution for 5 min, and the AP
color reaction was developed for 5–15 min according to thehcuge.ch.sprot/prosite.html/) and MOTIF (motif.genome.
ad.jp/), and search for sorting signals was done by psort. manufacturer’s specification.
Germ-line transformation and complementation of mu2 bynibb.ac.jp (Nakai et al. 1994).
Isolation of cDNAs: To isolate cDNAs from the mu2 region, the transgene: Three genomic fragments shown in Figure 1b
(E1, 10.3 kb; C2, 8.5 kb; A6c, 7.3 kb) were excised fromthree cDNA libraries were screened: a cDNA library from
adult male and female bodies (purchased from Novagen), an recombinant phages isolated from the genomic library and
ligated into the P-element vector CaSpeR 4. These constructsovarian cDNA library in lgt11 (provided by A. Spradling),
and a cDNA library from embryos 0–8 hr after egglay in the were usedfor P-element-mediatedtransformation. Transgenic
lines were obtained with insertions into chromosome II. TopNB40 plasmid vector (Nicholas Brown, Harvard University).
TheovariancDNAlibraryinlgt22A(Stroumbakisetal.1994) test for complementation of the mu2
a
phenotype, females of
the genotype w; P[w
1
];vemu2
a
were irradiated with 5 Gy ofwas only used for rapid amplification of cDNA ends (RACE)
analysis (see below). cDNA libraries were plated at 5 3 10
4
g-rays and mated to yw/y
2
sc Y males. All progeny, therefore,
1027The Drosophila mutator2 Gene
had pigmented eyes according to the presence of the
mutational events because they give rise to twin y and
transgene.TheX/0maleswerescored asy males.As described
f spots. As shown in Table 1, mu2
a
exhibited a 6-fold
by Mason et al. (1997), some of these males were probably
increase in the frequency of total spots compared with
aneuploid because they retain the base of the maternal X
the wild-type control and a 17-fold increase in the fre-
chromosome. The y mutants included y females and y
2
males.
Because y mutants cannot be scored when most of the X
quencyof twinspots. Totest fortheeffect ofirradiation,
chromosome is missing, the percentage of X/0 males is calcu-
third instar larvae were collected and irradiated with
lated as thenumber 3 100/total progeny, and thepercentage
5Gyofg-rays, which is the doseused toinduce terminal
of y mutants is calculated as the mutants 3 100/(mutants 1
deficiencies in mu2
a
oocytes. While this treatment in-
wild-type progeny).
creases the frequency of spots in wild-type females, it
has no discernible effect on the frequency of spots in
mu2
a
females. It is possible that the increase seen in
RESULTS
wildtype(0.4spotsperabdomen)istoosmalltoidentify
Somatic phenotype of mu2
a
: The mutator mu2
a
has
in mu2
a
abdomens, which already have nearly 5 spots
previously been characterized as having high mutation
per abdomen. When different isolates (alleles?) of mu2
rates in the female germ line (Mason et al. 1984, 1997).
were tested as homozygotes and heterozygotes with
To ask whether mu2
a
also has a somatic phenotype, two
mu2
a
, the results were in agreement with the homozy-
types of test were made. First, because of indications
gous mu2
a
results (data not shown).
thatDNArepair-defectivemutantsaresensitivetokilling
Mapping mu2 by deficiency breakpoints: To initiate
by chemical and physical agents (Baker et al. 1976a;
the cloning of the mu2 gene, we first refined our pre-
Boyd et al. 1987), larvae were tested for sensitivity to
vious deficiency mapping. Genetic mapping using a
methylmethane sulfonate (MMS) and g-rays. MMS sen-
large number of deficiencies has localized mu2 to the
sitivity was tested by crossing mu2
a
/TM3 males and fe-
cytological interval 62B11-C1 (Wang et al. 1994). Re-
males and treating larvae with 0.1% MMS according
cently, another gene, fs(3)ros, has been identified be-
to Boyd et al. (1976). In the untreated control cross,
tween the breakpoints Df(3L)Aprt66 and Df(3L)Aprt198
recoveryofmu2
a
progenywas72%relativetothehetero-
(D. Glover, personal commununication). We there-
zygous progeny (2 3 [54/149]) compared with 85% in
forereexaminated ourcollection ofdeficiencies(Wang
the treated set(2 3 [66/156]). Thisdose ofMMS killed
etal.1994)withbreakpoints inthe regionand identified
100% ofthe mei-41
A10
in acontrol cross.Similarly, adose
Df(3L)Aprt104, whichuncovers mu2but notfs(3)ros. The
of 60 Gy, which kills mei-41
D5
completely, has little effect
right (proximal) breakpoint of this deficiency was
on the relative survival of mu2
a
larvae (92% in the
mapped by genomic Southern blots to the 0.6-kb Pst
treated set). These experiments show that mu2
a
larvae
fragment just upstream of cDNA D (Figure 1B). This
and adults do not exhibit higher sensitivity to killing by
narrows the position of mu2 to the 6.3-kb region
DNA-damaging agents than does wild type. We have
between the breakpoints of Df(3L)Aprt66 and Df(3L)
also tested mu2
a
/Df and found no difference in mutant
Aprt104.Furtheranalysis ofcDNACas wellastheassign-
frequency between these and homozygous mu2
a
fe-
ment of fs(3)ros to one of these cDNAs will be reported
males.
elsewhere.
Second,the adult cuticlewas examinedfor theeffects
This region was subcloned in lambda phages from
of mu2
a
on mitotic recombination. Abdomens of y 1/
the 210-kb yeast artificial chromosome YAC N 23-77
1 f
36a
unirradiated females were examined for yellow
(Caietal.1994),whichspansthemu2locus.Theleftend
and forked spots according to the method of Baker et
ofthemolecularmapgeneratedfrom theseoverlapping
al. (1978). This assay can detect mutations at either of
phages is shown in Figure 1A. The positions of the
these two loci, deletions includingterminal deficiencies
right breakpoints of the deficiencies Df(3L)Aprt66 and
involving y, and mitotic recombination proximal to f.
These recombination events can be distinguished from Df(3L)Aprt198, which had been mapped by genomic
TABLE 1
Testing for effects of mu2
a
on mitotic recombination by scoring somatic spots
on abdomens of y 1/1 f
36a
females
g-Rays yellow forked Twin Total Spots per
Genotype (Gy) spots spots spots abdomens abdomen
1 0 53 36 11 118 0.85
1 5 59 52 21 105 1.26
mu2
a
0 214 123 153 99 4.0
mu2
a
5 176 143 143 100 4.6
mei-9
a
0 36 44 115 19 10.3
1028 A. Kasravi et al.
Figure 1.—Molecular map
of the mu2 genomic region.
(A) Contig of overlapping ge-
nomic lambda phages that
were subcloned from the yeast
artificial chromosome YAC N
23-77 (Wang et al. 1994).
BamHI sites are indicated by
vertical slashes, and the desig-
nations of the subcloned
BamHI fragments that were
used for screening cDNA li-
braries are indicated above the
restriction map. The broad
dashed lines indicate deleted
DNA in the four deficiencies
Df(3L)Aprt, which break in the
regionandextenddistally.The
phenotype of these deficien-
cies with respect to the two
known genes in the region,
mu2 and fs(3)ros, is indicated
abovethedashed linesforeach
deficiency. Also shown are the
positions of four transcription
units represented by the
cDNAs A–D, which were iso-
lated from cDNA libraries of
embryos and adults. By se-
quence homology, cDNA B
represents the gene for Dro-
sophila citric acid synthetase.
cDNA D is the mu2 transcript.
(B) Partial restriction map of
the mu2 gene region with posi-
tion of cDNAs C and D. The
PstI site at the proximal end of
the sequence shown in Figure
2 is marked with an asterisk.
Threegenomicfragmentsused
for germ line transformation
are indicated. B, BamHI; R,
EcoRI; X, XhoI; C, ClaI; P, PstI;
Xb, XbaI; S, SspI.
Southern blots (Wang et al. 1994), are now confirmed Probes from the proximal side of this region, around
the Df(3L)Aprt198 breakpoint, recognize two cDNAs,by cloning the genomic fragment containing the
breakpoints from recombinant phage libraries and by which overlap with their 39 noncoding regions by 83
bp.The 1.2-kbcDNA A, whichhas nosignificant homol-sequencing across the breakpoints (not shown). The
rightbreakpointofDf(3L)Aprt66wasfoundtobelocated ogy to any sequence in the database, lies outside
Df(3L)Aprt198. The 1.8-kb cDNA B represents the gene0.8 kb downstream of the 39 end of cDNA D, and the
right breakpoint of Df(3L)Aprt198 was located in the for 3-oxoacid-CoA-transferase (citric acid synthetase),
the key enzyme in the mitochondrial ATP-generatinggenomic BamHI fragment S7#9, within the gene repre-
sented by cDNA B (Figure 1B). Krebscycle, asshown bypartial sequencingand concep-
tual translation. The sequenced part of the DrosophilaIsolation of cDNAs: To identifytranscripts inthe mu2
region, three cDNA libraries were screened with sub- protein has 54–75% identityto thehomologous protein
from pig and somewhat less homology to the enzymecloned fragments fromthe genomic regionbetween the
right breakpoints of Df(3L)Aprt66 and Df(3L)Aprt198. from C. elegans and Bacillus subtilis (data not shown).
Two cDNAs lie at the other end of the probed region.Multiple isolates of each of the cDNAs were obtained.
Four different transcription units (termed A–D) were Preliminary sequencedata forcDNA C indicate thatthe
encoded protein has z25% identity to cardiac muscleidentified,and their positions on the genomic map were
determined by restriction analysis and partial sequenc- myosin over its entire length and to a large human
protein called mitosin, which is believed to be responsi-ing of the genomic and the corresponding cDNA frag-
ments (Figure 1A). ble for mitotic progression (Zhu et al. 1995).
1029The Drosophila mutator2 Gene
TABLE 2
pletely sequenced. Alignment with the corresponding
genomic region revealed nine small introns in the mu2
Complementation of mu2
a
by a transgene
gene, rangingfrom 51to 119 bpin length.The position
of the 39 end of the mu2 cDNA was confirmed by se-
Progeny
quence comparison to the genomic region. To deter-
Transgene
a
y
1
XO males (%) y mutants (%)
minethelocationofthe59 end,RACEexperimentswere
performedbyPCRwithtwodifferentcDNAlibraries:the
None 22453 656 (2.83) 75 (0.33)
P[w
1
]C2 7326 93 (1.25) 0
0–8-hr embryonic cDNA library (Nicholas Brown) and
P[w
1
]E1 12707 145 (1.13) 3 (0.02)
the ovarian cDNA library in lgt22A (Stroumbakis et
P[w
1
]A6c 10853 316 (2.82) 31 (0.28)
al. 1994). PCR was performed with an outward-facing
primer mu2cDNA-10, which is located 339 bp from the
w; P[w
1
];vemu2
a
were irradiated and crossed to yw/y
2
sc
59 end of the cDNA-26H9, and an inward-facing primer
Y males as described by Mason et al. (1997).
a
As shown in Figure 1B.
fromeitherthe NB40cloningvector orfromthe lgt22A
phage. Sequences of the genomic PstI-BamHI fragment
(see Figure 1B) upstream of cDNA D were used to posi-
Germ-line transformation: The position of mu2 was
tion the 59 end of the RACE products (Figure 2). All
confirmedby germ-linetransformation. Threegenomic
PCR fragments obtained from the embryonic cDNA li-
fragments, shown in Figure 1B, were tested for the abil-
brary byRACE terminatedat the sameposition (nt591)
ity to rescue the mu2
a
phenotype. In each case the
asthecDNA-26H9.ThelongestRACE productobtained
transgene had inserted into the second chromosome.
from the ovarian cDNA library in lgt22A terminated at
Following the assay described earlier (Mason et al.
an adenosine 45 bp further upstream (nt 546). Around
1997), w; P[w
1
];vemu2
a
females were treated with 5 Gy
both putative 59 termini there is a perfect match to the
of g-radiation and mated with yw/y
2
sc Y males for 24
arthropod pentanucleotide capsite TCAGT (Cherbas
hr, and the progeny were scored for y mutants. Results
and Cherbas 1993), suggesting thatboth positions may
are compiled in Table 2. Two fragments, C2 and E1,
be used as transcription start sites; it is not clear, how-
that both include the genomic region around cDNA D,
ever, whether the longer molecule may represent an
complemented mu2
a
completely. The third fragment,
ovary-specific transcript. A TATA-box-like sequence
A6c, which contains the region encompassing cDNA C
(boxed) is located at nt 496–502. The mu2 sequence is
and the 59 half of cDNA D, did not complement. Thus,
represented in five ESTs deposited into the databases
on the basis of its position between the breakpoints
by the Berkeley Drosophila Genome Project. The end-
of Df(3L)Aprt66 and Df(3L)Aprt104 and the phenotypic
points of the three longest 59 EST sequences are indi-
rescue by the genomic fragments C2 and E1, we con-
cated in Figure 2. Two of them (LD21703, GH15294)
clude that cDNA D represents the mu2 transcript.
are slightly shorter than our cDNA 26H9, the third one
Molecular structure of the mu2 gene: The longest
(LD10697) terminates 6 bases upstream of the end of
isolate of the mu2 cDNA (cDNA-26H9) is 4059 bp in
cDNA 26H9, but well within the longest cDNA deter-
length and was obtained from the 0–8 hr embryonic
mined by RACE. Taken together, these data define the
cDNA library. Shorter versions were also isolated from
position of the mu2 59 end.
The first potential ATG start codon that is in-framethe othercDNA libraries screened. This cDNAwas com-
Figure 2.—Nucleotide se-
quence of the 59 upstream geno-
mic region of the mu2 gene. The
PstI site at the proximal end of
the sequence is marked with an
asterisk in Figure 1. A potential
TATA motif is boxed, and two
capsite consensus sequences
(TCAGT) are underlined. The
first in-frame ATG start codon is
showninboldfacetype.Lowercase
sequences were determined from
RACE products of an ovarian
cDNAlibrary,extendingtont546,
and of a cDNA library from em-
bryos, extending to nt 591. The
longest isolate of a mu2 cDNA
from a0- to 8-hr embryonic cDNA
library (cDNA-26H9) alsoextends
to position 591 of this sequence.
The 59 termini of the threelongest EST sequencesgenerated by the Berkeley DrosophilaGenome Project, and their identification
numbers, are indicated (GenBank accession no. AF148494).
1030 A. Kasravi et al.
Figure 3.—Amino acid se-
quence of the MU2protein de-
duced from conceptual trans-
lation of mu2 cDNA-26H9. A
potential phosporylation site
forDNA-dependent proteinki-
nase is shaded. Three nuclear
localization signals are boxed.
Apotentialhelix-loop-helixdo-
main dimerization motif pre-
ceded by a basic region sug-
gesting DNA binding ability of
MU2 is underlined in boldface
betweenpositions193and250.
Five short degenerate amino
acid repeats of unknown func-
tion are also underlined.
with the long open reading frame on cDNA-26H9 is mu2 appears to encode a novel protein. No striking
homologies to any protein in the database were found.located only 24 or 69 bp, respectively, downstream of
the two putative transcription initiation sites, resulting The highest scoring protein was KIAA0170 (accession
no. D79992), which is an unidentified human proteinin a short59 nontranslatedleader sequence. Thispoten-
tial start codon is preceded by CAGG (see Figure 2), derived from conceptual translation of a cDNA isolated
from the myoblast cell line KG-1 (Nagase et al. 1996).a Drosophila start site consensus sequence (Cavener
1987). Conceptual translation of the mu2 cDNA-26H9 Homologies between the two proteins are predomi-
nantly located in the basic carboxy-half of the MU2indicates that the mu2 gene encodes a protein of 1261
aminoacids(Figure 3).Analysisofthe aminoaciddistri- protein, averaging z35%identity.Theshort amino acid
sequence KNRSS (1035–1039 in MU2; see Figure 3)bution pattern reveals a bipartite organization of the
protein. The amino-terminal half of the molecule occurs identically four times in KIAA0170 and another
five times in slightly modified form, contributing to the(amino acids 1–600) is acidic (pI. 4. 37), with a prepon-
derance of Asp 1 Glu (15.4%) over Lys 1 Arg (7.8%). homology between the two proteins. The function of
this motif, if any, is not known.The carboxy-terminal half of the MU2 protein is basic
(pI. 9. 87; Asp 1 Glu, 13.7%; Lys 1 Arg, 20.1%). A Developmental expression of mu2: On a develop-
mental Northern blot a single 4.0-kb transcript hybrid-search for functional motifs detected several potential
phosphorylation sites for various protein kinases. Of ized to the mu2 probe (Figure 4A). Its size is consistent
with the length of the cDNA-26H9. mu2 is expressedspecific functional significance may be the sequence
SQE in the amino half of the molecule, which is the at all developmental stages, and the transcript is most
abundant in adults and somewhat less so in early em-phosphorylation target motif for DNA-dependent pro-
tein kinase. This motif is found to be phosphorylated bryos and pupae. Lower levels are detected in late em-
bryos and inthe larval instars. Expression levels inadultwith high efficiency in human p53 (Anderson 1993).
Threepotentialnucleartargetingsites(boxed)arepres- females are not significantly higher than in adult males
(Figure 4B).ent in the carboxy-half of the protein, suggesting nu-
clear localization with a probability of 0.7 (Nakai et al. The mu2 transcript was localized in various tissues by
whole-mount in situ hybridization with a digoxigenin-1994). The signature pattern of PROSITE modeled to
detect the second amphipathic helix of myc-type helix- labeled RNAprobe generated fromthe mu2cDNA (Fig-
ure 5). In all cases, only the antisense probe gave aloop-helix domains recognizes the region between posi-
tions235and 243.Thisdomainmediates proteindimer- positive reaction. We first studied mu2 expression in the
female germ line. Within an ovary, each Drosophilaization and is often preceded by a short basic region in
DNA-binding transcription factors. Such a basic region ovariole contains egg chambers at different stages of
maturity, beginning with the germarium at the anteriorcontaining two lysine residues is also present on MU2
between positions 193 and 203 before the potential he- end and followed by cysts in stages 2 to 14 (King 1970;
Spradling1993). A stage14 oocyteis matureand readylix-loop-helix domain from position 204–250 (bold
underline), suggesting possible DNA binding capability for fertilization. In the germarium, the oocyte, which
resides at the posterior end of the 16-cell egg chamber,of MU2. Another feature of the protein is five slightly
degenerate amino acid repeats between positions 412 initiates meiosis, while the remaining 15 cells develop
into nursecells, undergoendoreplication of theirDNA,and 522 (underlined) whose significance is unknown
at this time. and become transcriptionally active. RNA molecules
1031The Drosophila mutator2 Gene
Figure 4.—Northern blots
of poly(A)
1
RNA from various
developmental stages hybrid-
izedtoa
32
P-labeledmu2probe.
(A) A 4.0-kb mu2 transcript is
detected at all developmental
stages and is most abundant in
adult flies. Less mu2 RNA is
present in 0- to 12-hr-old em-
bryos and in pupae, and lower
levelsof mu2 mRNA aredetect-
ablein12-to24-hr-oldembryos
and in the larval instars. (B)
Hybridization of the same
Northern blot as in A with a
b-tubulin probe to control for
RNA loading. (C) Comparison
of mu2 RNA levels in poly(A)
1
RNA from adult females and
males; and (D) hybridization
ofthesameNorthernblot asin
(C) withthe ribosomal protein
RP-49 probe to control for
RNA loading.
and proteins synthesized by the nurse cells are trans- presumably the oocyte. At stages 5 to 6, the transcript
becomes transiently more concentrated atthe posteriorported into the oocyte via the ring canals. The bulk of
the RNA molecules is released from the nurse cells into margin of the oocyte, but after stage 8–9 it accumulates
at the anterior end of the oocyte, where it appears tothe oocyte at stage 11. Hybridizing ovarioles with the
mu2 antisense probe confirmed that the mu2 gene is form a ring-shaped pattern. This pattern of RNA accu-
mulationanddistribution isreminiscentofother mater-strongly expressed during oogenesis (Figure 5A). The
transcript can already be detected in the germarium, nal RNA transcripts that are deposited early into the
oocyte and become localized at the anterior margin ofwhere it appears in only one cell of the young oocysts,
Figure 5.—(A) Whole-
mount in situ hybridization
of digoxigenin-labeled mu2
antisenseRNAprobetoova-
ries, where the transcript
can already be detected in
thegermarium.Instage8–9
cysts, it accumulates at the
anterior end of the oocyte,
where it appears to form a
ring-shapedpattern.(B)No
MU2 transcript is present in
testes, except in thesomatic
sheath cells. MU2 RNA is
alsodetected(C)inthepro-
liferating zonesof the larval
brainand (D) in thecycling
cells of the morphogenetic
furrow of the eye disc. (E)
MU2 RNA is ubiquitous in
cycle8embryos,whereitap-
pears to be more abundant
in the anterior half. (F) In
later embryonic stages, mu2
is expressed in the prolifer-
ating nervous system.
1032 A. Kasravi et al.
the oocyte, e.g., the transcript of the axis-determining Our genetic analyses of the mu2
a
allele have estab-
lished that functions for MU2 exist in the female germtranscription factor bicoid (Berleth et al. 1988; St.
Johnston et al. 1989). No MU2 transcript was detected line as well as in somatic tissues. The developmental
expression pattern and the transcript localization arein testes, except in the somatic sheath cells (Figure 5B).
MU2 RNA is also present in larval somatic cells, espe- consistent with these phenotypes. In ovaries, MU2 RNA
has an unusual pattern of localizationin that it accumu-cially in proliferating tissues such as the brain (Figure
5C) and imaginal discs (Figure 5D). Interestingly, mu2 lates in the oocyte of very young ovarian cysts in the
germarium and at stage 8–9 MU2 RNA becomes local-expression seems to be increased in the proliferating
zones of the larval brain (Figure 5C) and in the cycling izedattheanteriorendoftheoocyte.Whilethemajority
of maternal RNAs are deposited into the oocyte at stagecells of the morphogenetic furrow of the eye disc (Fig-
ure 5D). Terminally differentiated tissues such as the 11 when the nurse cells inject their contents into the
growing oocyte, a few RNA species are transported intolarval salivary glands do not express mu2 (not shown).
MU2 RNA is ubiquitous in early embryos, indicating a theoocytemuchearlier,wheretheybecomelocalizedto
specific regions (Mahajan-Miklos and Cooley 1994).maternal contribution to theegg. Beforenuclear migra-
tion, the RNA appears to be more abundant in the These RNAs include anterior-located transcripts encod-
ing the axis-determining transcription factors BICOIDanterior half and concentrated in the energids sur-
rounding each nucleus (Figure 5E). In later embryonic (Berleth et al. 1988; St. Johnston et al. 1989) and
Fs(1)K10 (Cheung et al. 1992), as well as transcripts ofstages, mu2 seems to be expressed in the proliferating
nervous system (Figure 5F). genes thatencode structural components ofthe oocyte,
suchas possiblecytoskeletal components,Adducin-HTS
(Yue and Spradling 1992; Ding et al. 1993), BIC-D
DISCUSSION
(Suter et al. 1989), a chromatin condensation protein,
BJ1 (Frasch 1991), RNA-binding proteins, ORBWe have cloned and sequenced the D. melanogaster
mu2 gene. Conceptual translation of its cDNA shows (Lantz et al. 1992), and a DNA-binding factor, YEM-a
(Ait-Ahmed et al.1987). RNA localization inthe oocytethat it encodes a novel 139-kd polypeptide that is very
likely targeted to the nucleus. A nuclear localization is dependent on the organization of the cytoskeleton
(Pokrywka and Stephenson 1991; Theurkauf et al.would be consistent with the mu2
a
phenotype, but con-
firmation has to await the production of antibodies to 1992, 1993;Lane andKalderon 1994).In fact,the shift
of polarity of the microtubules in the oocyte duringtheMU2protein. Analysisoftheamino aciddistribution
pattern reveals a bipartite organization of the protein: stages 7 to 8 (Theurkauf et al. 1992) appears to be
responsible for the shift from the accumulation of ma-the amino-terminal half of the molecule (amino acids
1–600) is acidic, while the carboxy-terminal half of the ternal RNAs from the posterior end of the oocyte to
the accumulation at the anterior end. It has been pro-MU2 protein is basic. The presence of three nuclear
localization signals and a myc-type helix-loop-helix do- posed that anteriorly localized RNAs are actively trans-
ported along the microtubules by minus end-directedmain preceded by a short basic region suggest nuclear
localization and possible DNA-binding capability of motors (Theurkauf et al.1992, 1993),which would also
account for the rapid transport from the nurse cellsMU2.
Previous genetic data (Mason et al. 1997) suggested through the ring canals. In all of these aspects, MU2
RNA localization resembles that of bicoid RNA. Onethat, whilemu2 functionis importantfor repairof radia-
tion-induced damage in oocyte chromosomes, it is not possible reason for this anterior localization of MU2
RNA may be that the MU2 protein is needed at thean integral component of DNA repair per se, because
irradiated sperm chromosomes are repaired normally anterior end of the oocyte, where the oocyte nucleus
resides. MU2 protein may enter the oocyte nucleus toin a mu2
a
cytoplasm. mu2 may also play a role in meiotic
recombination because the mu2
a
mutant exhibits a sig- perform its function.
How can the somatic and germ-line phenotypes ofnificantdecreasein meioticrecombination(Masonand
Champion1989). We reporthere onthe somaticeffects mu2 be explained? We propose that mu2
a
is defective
in an ancillary process to DNA repair in oocytes and inof mu2
a
: in an assay for somatic mutation and recombi-
nation (Baker et al. 1978), mutant females showed a zygotes produced by mu2
a
mothers. Three observations
suggestthatmu2mutantsdelayordisrupttheprocessingsignificantincrease inspontaneousrecombination.Sim-
ilar differential effects on meiotic and mitotic recombi- of DSBs in mature oocytes and of recombination inter-
mediates in early oocytes. First, in irradiated wild-typenation have been described for DNA repair-defective
mutants, such as mei-9
a
. We have not tested for the females, chromosomal breaks have a half-life of only
10–20 min (Wu
¨
rgler and Matter 1968) and are effi-occurrence of somatic spots in mutant males, because
the standard procedure for this test uses the mwh and ciently repaired while the oocyte is waiting for fertiliza-
tion. By contrast, breaks induced in mu2
a
oocytes haveflr
3
markers on 3L. mu2 may be within a map unit of
mwh, and given the phenotypes involved, a mwh mu2 a half-life of z20 hr (Mason et al. 1997). Second, mu2
a
reduces meiotic recombination by 25% and increasesdouble mutant chromosomewould be difficultto make.
1033The Drosophila mutator2 Gene
meiotic chromosome malsegregation fivefold (Mason of only S and M phase (Raff and Glover 1988). Cou-
pling to a mitotic cell cycle checkpoint occurs after theand Champion 1989). Third, while mu2
a
has only a
slight effect on the fertility of unirradiated females introduction of a G
2
phase in cell cycle 14 following
cellularization,whichisregulatedattheG
2
/Mboundary(Mason and Champion 1989), combining mu2
a
with
weakallelesofmei-41,whichencodesaDrosophilaATM- by the string phosphatase (Edgar and O’Farrell 1989,
1990). After nuclear migration, the syncytial embryorelated kinase and may control a DNA damage-respon-
sivecellcyclecheckpoint(Harietal.1995),dramatically deals with division errors by eliminating nuclei con-
taining abnormal products from the subcortical cyto-decreases female fertility (J. M. Mason, unpublished
observation).This isconsistent withthe notionthatmu2 plasm and delaying the initiation of anaphase
(Fogarty et al. 1997; Sullivan et al. 1990, 1993). Thus,mutants have difficulties processing meioticrecombina-
tionintermediatesand delayprogressionthroughmeio- unrepaired breaksinducedinmu2 postrecombinational
oocytes will not cause arrest until after nuclear migra-sis until the recombination intermediates are resolved.
The mei-41 mutation may allow the cell to progress tion. Therefore, shortly after fertilization of the egg,
these breaks can become capped by a yet unknownthrough meiosis without completing recombination
and thus deposits recombination intermediates, possi- protein, resulting in fixation as terminal deficiencies.
This capping pathway may be very active in the syncytialble chromosome fragments, in the zygote, where they
are lethal. Therefore, mei-41; mu2 cells can progress embryo, whichmay containhigh levels of such aprotein
to cap the large number of new telomeres generatedthrough meiosis before completing recombination, re-
sulting in embryonic death. in the early rapid S–M cycles.
On the other hand, mu2
a
has no discernible effect in
Wethankthefollowingpeoplefortheircontributionstotheproject:
the male germ line (Mason et al. 1997). In Drosophila,
AlanSpradling,KavitaArora,andMark Stapeleton for cDNA libraries;
Tibor To
¨
ro
¨
k for help with the Northern blots; Mike Boedigheimer
lesions induced in mature sperm by irradiating mu2
a
for the RP-49 probe; and Heidi Theisen for advice with the tissue in
males are normally repaired after fertilization (Muller
situhybridizations.Thefollowingundergraduatestudentshelpedwith
1940; Maddern and Leigh 1976) under the genetic
the project at UCI: Hung Tran, Dan Le, Sandy Chuang, and Frances
control of the female (Graf et al. 1979). Irradiation of
Kobeski. We thank Kevin Lewis, Elena Kurenova, and Ali Haoudi for
mu2
a
males does not result in terminal deficiencies,
critical reading of the manuscript. S.B. thanks the Association de
la Recherche contre le Cancer and the Training and Mobility of
increased mutation, or decreased fertility, even when
Researchers Programme from the European Union for Post-Doctoral
the irradiated males are crossed to mu2
a
females. These
fellowships. S.B. acknowledges David Glover for his interest and sup-
results suggest that mu2 does not encode a repair en-
port throughout this work, which is funded through grants from the
zyme and that the general DNA repair machinery is
Cancer Research Campaign of Great Britain.
not defective in mu2
a
females. mu2 RNA expression in
ovaries but not in testes is consistent with the observed
sex-specific phenotype in the germ line. Before the first
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