From the Department of Zoology and Genetics, Iowa State University,
Ames, Iowa 50011
Received for publication, December 30, 2002, and in revised form, January 20, 2003
Using a yeast two-hybrid screen we have
identified a novel isoform of the lola locus, Lola zf5,
that interacts with the chromosomal kinase JIL-1. We characterized the
lola locus and provide evidence that it is a complex locus
from which at least 17 different splice variants are likely to be
generated. Fifteen of these each have a different zinc finger
domain, whereas two are without. This potential for expression
of multiple gene products suggests that they serve diverse functional
roles in different developmental contexts. By Northern and Western blot
analyses we demonstrate that the expression of Lola zf5 is
developmentally regulated and that it is restricted to early
embryogenesis. Immunocytochemical labeling with a Lola zf5-specific
antibody of Drosophila embryos indicates that Lola zf5 is
localized to nuclei. Furthermore, by creating double-mutant flies we
show that a reduction of Lola protein levels resulting from mutations
in the lola locus acts as a dominant modifier of a
hypomorphic JIL-1 allele leading to an increase in
embryonic viability. Thus, genetic interaction assays provide direct
evidence that gene products from the lola locus function
within the same pathway as the chromosomal kinase JIL-1.
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INTRODUCTION |
Chromatin structure as well as the differential expression of
transcription factors plays an important role in the regulation of gene
expression (1-3). We have recently identified a chromosomal tandem
kinase, JIL-1, that modulates chromatin structure in
Drosophila (4-6). JIL-1 is an essential kinase, and in
JIL-1 nulls and hypomorphs euchromatic regions of chromosomes are
severely reduced and the chromosome arms are condensed (6). These
changes are correlated with decreased levels of histone H3
Ser-10 phosphorylation (6). JIL-1 has been implicated in
transcriptional regulation, as it localizes to the gene-active
interband regions of interphase larval polytene chromosomes (4) and has
been found to associate with at least one chromatin-remodeling complex,
the male specific lethal (MSL)1 dosage compensation
complex (5). The MSL complex is required for the necessary
hypertranscription of genes on the male X chromosome for dosage
compensation in flies (reviewed in Ref. 7). This enhanced transcription
is thought to arise from MSL complex-induced histone H4 acetylation
generating a more open chromatin structure (8). The increased histone
H3 Ser-10 phosphorylation levels that JIL-1 promotes on the male X may
also play a role in maintaining a more open and active chromatin
structure (5, 6). However, it is not known whether physiological
substrates of JIL-1 may include other proteins such as transcription
factors or whether there are proteins directly regulating the function
of JIL-1. To identify proteins that interact with JIL-1 we carried out
yeast two-hybrid screens using different JIL-1 regions as baits. Here we report that a novel splice form from the lola locus,
which we have named Lola zf5, was identified in such a screen to
interact with the first kinase domain (KDI) of JIL-1.
The lola locus was first characterized as a mutation
affecting longitudinal axon growth within the central nervous system (9). Two isoforms from the lola locus have been previously described, Lola long and Lola short (10). Lola long contains two zinc
finger motifs and is a transcription factor with DNA binding activity
(10, 11). The lola locus mediates decreased copia
retrotransposon mRNA expression in the central nervous system while
upregulating its expression in gonads (11). In addition, lola is required for proper expression of the axonal
guidance proteins Robo and slit in the central nervous system (12).
Although both Lola isoforms share a BTB/POZ domain (13-15), Lola short
contains no zinc finger domains (10). BTB domains are known to mediate dimerization (16, 17), which includes the ability to promote heterophilic interactions of different BTB domain-containing isoforms (15, 18, 19). BTB domain-containing zinc finger proteins have been
strongly implicated in regulation of chromatin structure and gene
expression (20). For example, human B cell lymphoma (BCL-6) and
promyelocytic leukemia zinc finger oncoproteins have been shown to act
as transcriptional repressors (21-23). Specific recruitment of
repressor complexes to target promoters occurs through binding of
corepressors to the hydrophobic BTB dimer pocket (24). Corepressor
binding recruits a complex containing a histone deacetylase (25, 26)
that represses transcription by inducing condensed chromatin
architecture (reviewed in Ref. 27).
In this study we characterized the genomic organization of the
lola locus and show that it is a complex locus from which at least 17 different splice variants are likely to be transcribed. All
isoforms from the lola locus share a common BTB domain,
whereas 15 of the splice variants each contain a different zinc finger domain. We show that one of these variants, Lola zf5, physically interacts with the JIL-1 kinase and that its expression is
developmentally restricted to early embryogenesis. Furthermore, we show
that a P element insertion in the lola locus enhances the
viability of a hypomorphic JIL-1 allele, indicating opposing functions
of the JIL-1 and lola loci. Although only Lola
long and Lola short have so far been studied in detail, it has long
been known that P insertion mutations in lola that prevent
the expression of some if not all the isoforms have very complex
phenotypes (10-12, 28). Our findings suggest that this complexity may
derive from the expression pattern of multiple gene products from the
lola locus that are likely to serve diverse functional roles
in different cellular and developmental contexts.
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MATERIALS AND METHODS |
Drosophila Stocks--
Fly stocks were maintained according to
standard protocols (29). Oregon-R was used for wild type preparations.
JIL-1EP(3)3657 and JIL-1z2
alleles have been previously described (6). Balancer chromosomes and
mutant alleles are described in (30). The lola00642
mutant stock cn1
P{ry+t7.2 = PZ}lola00642/CyO;
ry506 was obtained from the Bloomington
Drosophila Stock Center. Hatch rates were determined by
counting the number of eggs laid on standard apple juice/agar plates
and then counting the number of unhatched eggs at 22 h and again
at 48 h after egg laying. All genetic crosses and interaction
assays were conducted at 23 °C.
Identification and Molecular Characterization of Lola
zf5--
JIL-1 cDNA sequence encoding a 304-amino acid fragment
(Tyr251-Glu554) comprising the first
kinase domain (JIL-1 KDI) of JIL-1 was subcloned in-frame into the
yeast two-hybrid bait vector pGBKT7 (Clontech)
using standard methods (31) and verified by sequencing (Iowa State
University Sequencing Facility). The JIL-1 KDI bait was used to screen
the Clontech MatchmakerTM 0-21 h
embryonic Canton-S yeast two-hybrid cDNA library according to the
manufacturer's instructions. A positive cDNA clone KDIJ1 was
isolated, retransformed into yeast cells containing the JIL-1 KDI bait
to verify the interaction, and sequenced. Homology searches identified
SW59 (GenBankTM Z97377) as well as the
lola locus. We obtained the SW59 cDNA from Dr. D. Zhao
(University of Edinburgh) and lola ESTs (LD28033, LD33478,
and LD17361) from ResGen Invitrogen and assembled the full-length Lola
zf5 coding sequence. We note a few differences between Z97377 and the
Lola zf5 full-length cDNA assembled in this study: 1) 62 nucleotides at the most 5'-end of Z97377 may be a library construction
artifact, because they are not present in other Lola zf5 EST clones or
the reported lola genomic sequence (32); 2) six gaps are
present between Z97377 and our KDIJ1 fragment. At all of the gaps,
KDIJ1 cDNA sequence is 100% identical to the available ESTs and
genomic sequence (33, 32). Sequencing of EST clones LD28033 and LD33478
support the presence of two Lola zf5 splice isoforms using alternative
5'-UTR sequences (5'-a and 5'-c, respectively) but we have not
confirmed use of 5'-b and 5'-d UTR alternative exons for Lola zf5, as
predicted in the November 30th, 2002 genome project update.
Antibody Generation and Antibody Affinity
Purification--
Rabbit anti-Lola common region polyclonal antibody
was a generous gift of Dr. Edward Giniger and has been
previously characterized (10). Hope and Odin rabbit anti-JIL-1
polyclonal antibodies were described in Jin et al.
(4). Affinity purification of anti-Lola and anti-JIL-1 polyclonal
antibodies was as described in Giniger et al. (10) using
lacZ-Lola and GST-JIL-1 fusion proteins, respectively. To generate Lola
zf5-specific monoclonal antibody (mAb) 7F1, the KDIJ1 cDNA fragment
(encoding Lola zf5 amino acid residues 427-748) was cloned in the
correct reading frame into pGEX4T-1 (Amersham Biosciences), verified by
sequencing, and GST-zf5 fusion protein was induced in
Escherichia coli according to standard protocols
(Amersham Biosciences). Injection of GST-zf5 fusion protein into BALB/C
mice, and generation of monoclonal hybridoma lines was performed
by the Iowa State University Hybridoma Facility according to standard
protocols (34).
Immunohistochemistry--
Embryos were dechorionated in 50%
Chlorox solution, washed with 0.7 M NaCl/0.2% Triton X-100
and fixed in a 1:1 heptane:fixative mixture for 20 min with vigorous
shaking at room temperature. The fixative was either 4%
paraformaldehyde in phosphate-buffered saline (PBS) or Bouin's Fluid
(0.66% picric acid, 9.5% formalin, 4.7% acetic acid). Vitelline
membranes were then removed by shaking embryos in heptane-methanol (35)
at room temperature for 30 s. Embryos were blocked in PBS with 1%
normal goat serum (Cappel) and 0.4% Triton X-100 and incubated
overnight in mAb 7F1 primary antibody diluted in blocking buffer.
Embryos were washed in PBS with 0.4% Triton X-100, incubated for
2.5 h with TRITC-conjugated goat anti-mouse secondary antibody
(1:200) (Cappell), washed in PBS with 0.4% Triton X-100 followed by a
PBS-only wash. For visualization of DNA the antibody-labeled embryos
were incubated in 0.2 µg/ml Hoechst 33258 (Molecular Probes) in PBS
for 10 min. The final preparations were mounted in glycerol with 5%
n-propyl gallate and viewed with a 40× NeoFluor objective
on a Zeiss Axioskop equipped with filter sets optimized and selective
for rhodamine and UV detection. Digital images were obtained using a
Spot-cooled charge-coupled device camera (Diagnostic Instruments).
Northern and Western Blot Analysis--
Approximately 1 g
of wild type Oregon-R animals from different stages was collected and
ground under liquid nitrogen, and total mRNA was purified using the
Poly(A)+ mRNA purification kit (Ambion). 5 µg of
poly(A)+ mRNA from each stage was fractionated on 1%
agarose formaldehyde gels, transferred to Duralon-UVTM
nylon membrane (Stratagene), and hybridized with
32P-labeled probe overnight at 65 °C according to
standard high stringency protocols (31). Lola zf5 isoform-specific
probes were generated by purifying 1.8 kb of unique 3'-end Lola zf5
cDNA sequence using a QiaQuick gel extraction kit (Qiagen) and
synthesizing random primer 32P-labeled probe using the
Prime-A-Gene kit (Promega) according to the manufacturer's
instructions. As a loading control, a cDNA fragment of RP49
(ribosomal protein L32) (36) was PCR-amplified from the
Clontech yeast two-hybrid cDNA library
described above and confirmed by sequencing. After stripping the Lola
zf5 signal, labeled RP49 cDNA was used to probe the same membrane
to normalize mRNA loading levels.
Protein extracts were prepared from staged dechorionated embryos,
larvae, pupae, or adults that were homogenized in
immunoprecipitation buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA,
0.1% Triton X-100, 0.1% Nonidet P-40, 2 mM
Na3VO4, pH 8.0) with added protease inhibitors
1.5 µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride (Sigma). Proteins were boiled in SDS-PAGE buffer, separated on
SDS-PAGE gels, transferred to nitrocellulose, blocked in 5% Blotto,
and incubated with anti-JIL-1 or anti-Lola antibody overnight. Blots
were then washed three times for 10 min in TBST (0.9% NaCl, 100 mM Tris, pH 7.5, 0.2% Tween 20), incubated with
horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse
secondary antibody (1:3000) (Bio-Rad) for 1 h at room temperature,
washed in TBST, and the antibody signal was detected with the ECL
chemiluminescence kit according to manufacturer's instructions
(Amersham Biosciences).
In Vitro Protein Interaction and Co-immunoprecipitation
Assays--
Approximately 5 µg of GST-KDIJ1 (Lola zf5 C terminus)
fusion protein or GST protein alone was coupled to glutathione-agarose beads (Sigma) and incubated with 0.5 ml of S2 cell lysates (3 × 106 cells) overnight at 4 °C. The beads were pelleted at
low speed and washed three times with 1 ml of immunoprecipitation
buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 0.1%
Nonidet P-40, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1.5 µg of
aprotinin, pH 8.0) for 10 min at 4 °C. The proteins retained on the
beads were analyzed by Western blot analysis using affinity-purified
Hope anti-JIL-1 polyclonal antibody.
For co-immunoprecipitation experiments, anti-JIL-1, anti-Lola zf5, or
control normal rabbit antibodies were coupled to protein G beads as
follows: 30 µl of Odin anti-JIL-1 serum, 30 µl of normal rabbit
control serum, or 1 ml of 7F1 hybridoma supernatant was coupled to 25 µl of protein G-Sepharose beads (Amersham Biosciences) for 2 h
at 4 °C on a rotating wheel in 50 µl of immunoprecipitation buffer. The appropriate antibody-coupled beads were incubated overnight
at 4 °C with 300 µl of 0-6 h embryonic lysate on a rotating wheel. Beads were washed four times for 10 min each with 1 ml of
immunoprecipitation buffer with low speed pelleting of beads between
washes. The resulting bead-bound immunocomplexes were analyzed by
SDS-PAGE and Western blotting according to standard techniques as
described in Jin et al. (4) using Hope antibody to detect
JIL-1, mAb 7F1 to detect Lola zf5, or Lola polyclonal antisera against
the Lola common core domain (10) to detect all Lola isoforms.
Bioinformatics--
Lola genomic DNA sequence corresponding to
nucleotides 118,125-182,622 of Drosophila melanogaster
genomic scaffold AE003829.3 (GI: 21627529, updated on September 20, 2002) (32) was used to search for zinc finger motifs. Consensus zinc
finger motifs include CXXC, HXXXXC, and
HXXXXH (X represents any amino acid) using
the "Find" function under "Edit" menu of Microsoft Word 98 software. In most cases, when an open reading frame (ORF) contained two
of the three consensus sequences, it was considered a potential zinc
finger motif and further analyzed. PCR primers were designed to amplify
Lola sequences containing the putative cDNA fragments specific to
the predicted zinc finger motifs using standard PCR protocols (31). The
forward PCR primer ZnF5P (5'-GGATGAACTTGGACTAATGGC-3') consists of Lola
common core sense sequence derived from exon IV, whereas reverse PCR
primers were designed to be isoform-specific, with individual reverse
primers comprised of antisense sequence based on the 3'-end of each
separate predicted zinc finger motif. Oligonucleotides were
designed using the Oligo 5.0 program. cDNA templates for PCR
reactions were from the 0- to 21-h cDNA MatchmakerTM
yeast two-hybrid library (Clontech) or a 3- to 12-h
cDNA library (Stratagene). PCR conditions were optimized for each
primer pair, respectively, and PCR products were directly sequenced.
The lola genomic sequence was also used to search for
homologs in the Drosophila EST databases with FLYBLAST
(available at www.fruit fly.org/blast/) (33) and with the Geneseqer
program (available at
bioinformatics.iastate.edu/cgi-bin/gs.cgi) (37). Returned
ESTs were compared with lola genomic DNA sequence using BLAST2 (available at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) at
NCBI (National Center for Biotechnology Information) (38). Putative
zinc finger motifs were aligned using Clustal W available at
www2.ebi.ac.uk/CLUSTALW (39).
 |
RESULTS |
The JIL-1 Kinase Interacts with a Novel Isoform of the lola
Locus--
To identify proteins that have direct interactions with the
JIL-1 kinase, we performed a yeast two-hybrid screen of a Canton-S 21-h
embryonic library using the first kinase domain of JIL-1 as bait. True
positive clones detected in the primary screen were confirmed by
-galactosidase two-hybrid interaction assays on filter paper
following retransformation of the candidate clones and JIL-1 KDI bait
plasmid into the yeast strain AH109 (data not shown). One of the
positive clones identified in this way was sequenced, and searches of
the Drosophila genome database revealed it to be the
COOH-terminal domain of a novel splice variant from the lola
locus, which we have named Lola zf5. Subsequently, the full-length
cDNA sequence for Lola zf5 was assembled from overlapping ESTs
obtained from the Drosophila genome project and is currently available as AY058586 as well as the SW59 clone (Z97377). Fig.
1A shows the amino acid
sequence of the predicted open reading frame of a protein of 748 residues with a calculated molecular mass of 79.4 kDa. The BTB domain
and zinc finger domains are underlined and boxed,
respectively. To further characterize the protein, a Lola zf5-specific
monoclonal antibody, 7F1, was generated against a GST fusion protein
containing the COOH-terminal region unique to Lola zf5. On immunoblots
of embryo protein extracts (0-6 h) mAb 7F1 detects Lola zf5 as a
single band migrating at 105 kDa (Fig. 1B). The Lola zf5
protein is highly acidic with a pI of 5.54 accounting for its anomalous
gel migration. Immunocytochemical labeling of early
Drosophila embryos with mAb 7F1 revealed the Lola zf5
protein to be localized to nuclei in a pattern similar to that obtained
by Hoechst labeling (Fig. 1, C and D).

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Fig. 1.
The predicted sequence of the Lola zf5
isoform and Lola zf5 antibody labeling. A, the complete
predicted amino acid sequence of Lola zf5. Lola zf5 is a 748-residue
protein with a calculated molecular mass of 79.4 kDa. The BTB domain is
underlined, and the zinc finger domains are
boxed. B, immunoblot of SDS-PAGE fractionated 0- to 6-h embryo extracts labeled with the Lola zf5-specific mAb, 7F1. The
antibody recognizes a single band migrating at 105 kDa. The migration
of molecular mass markers in kilodaltons is indicated to the
left. C and D, double labeling of a
syncytial embryo with mAb 7F1 and Hoechst. The labeling of mAb 7F1
(C) shows that Lola zf5 is localized in a pattern
overlapping with that of the Hoechst labeled nuclei
(D).
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To further explore the interaction between Lola zf5 and JIL-1 that we
observed in the yeast two-hybrid assays we performed pull-down assays
with the Lola zf5 COOH-terminal GST fusion protein using protein
extracts from the S2 cell line. The Lola zf5-GST fusion protein or a
GST-only control were coupled with glutathione-agarose beads, incubated
with S2 cell lysate, washed, fractionated by SDS-PAGE, and analyzed by
immunoblot analysis using JIL-specific antibody (Fig.
2A). Whereas the GST-only
control showed no pull-down activity, Lola zf5-GST was able to pull
down JIL-1 as detected by the JIL-1 antibody. In addition, we performed
co-immunoprecipitation experiments using embryonic lysates. For these
immunoprecipitation experiments, proteins were extracted from 0-6 h
embryos, immunoprecipitated using either JIL-1- or Lola zf5-specific
antibodies, fractionated on SDS-PAGE after the immunoprecipitation,
immunoblotted, and probed with antibodies to Lola zf5 and JIL-1,
respectively. Fig. 2B shows an immunoprecipitation
experiment using Lola zf5 antibody where the immunoprecipitate is
detected by JIL-1 antibody as a 160-kDa band that is also present in
the embryo lysate. This band was not present in lanes where
immunobeads only were used for the immunoprecipitation. Fig.
2C shows the converse experiment: JIL-1 antibody
immunoprecipitated a 105-kDa band detected by Lola zf5 antibody that
was also present in embryo lysate but not in control
immunoprecipitations with immunobeads only. These results strongly
indicate that Lola zf5 and the JIL-1 kinase are present in the same
protein complex.

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Fig. 2.
Lola zf5 and JIL-1 pull-down and
immunoprecipitation experiments. A, S2 cell lysate
incubated with a Lola zf5-GST fusion construct or a GST only control
was pelleted with glutathione-agarose beads and the interacting
protein(s) fractionated by SDS-PAGE, Western blotted, and probed with
JIL-1 antibody. Non-incubated S2 cell lysate was included as a control
(lane 1). The Lola zf5-GST construct was able to pull down
the 160-kDa JIL-1 protein as indicated by detection with JIL-1 antibody
(lane 2), whereas no interaction was observed with the GST
only control (lane 3). B, immunoprecipitation of
lysates from 0- to 6-h embryos was performed using Lola zf5 antibody
(mAb 7F1) coupled to immunobeads (lane 2) or with
immunobeads only as a control (lane 3) and analyzed by
SDS-PAGE and Western blotting using JIL-1 antibody for detection. JIL-1
is detected as a 160-kDa band in embryo extracts (lane 1) as
well as in the Lola zf5 antibody immunoprecipitation sample (lane
2) but not in the control sample (lane 3).
C, immunoprecipitation of lysates from 0- to 6-h embryos
were performed using JIL-1 antibody coupled to immunobeads (lane
2) or with immunobeads only as a control (lane 3) and
analyzed by SDS-PAGE and Western blotting using Lola zf5 antibody for
detection. Lola zf5 is detected as a 105-kDa band in embryo extracts
(lane 1) as well as in the JIL-1 antibody
immunoprecipitation sample (lane 2) but not in the control
sample (lane 3).
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The Complex lola Locus Encodes Multiple BTB Domain-containing
Proteins Each with Different Zinc finger Motifs--
Two alternatively
spliced isoforms of lola, Lola long and Lola short, have
been previously characterized (10). Lola long is a sequence-specific
DNA-binding protein with C2HC and
C2H2 zinc finger motifs, whereas Lola short
only has a very short COOH-terminal tail segment without zinc finger
motifs (10, 11). Fig. 3A shows
the domain structure of these two isoforms as compared with Lola zf5.
Lola zf5 has an NH2-terminal common region shared by all
cloned Lola isoforms that is followed by an isoform-specific COOH-terminal region (Fig. 3A). The Lola common region is
encoded by four exons and contains a 120-amino acid
NH2-terminal BTB domain as well as a nuclear localization
signal (Fig. 3, A and B). The COOH-terminal
domain of Lola zf5 contains tandem C2HC and
C2H2 zinc finger motifs (Fig. 3A).
The position of the Lola zf5 zinc fingers are very close to the common
region in contrast to the Lola long isoform in which the zinc fingers
are positioned at the COOH-terminal end.

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Fig. 3.
Comparison of Lola isoforms.
A, schematic diagrams of the domain organization of Lola zf5
and the two Lola isoforms, Lola long and Lola short, drawn to scale.
Each Lola isoform shares a common region consisting of an
NH2-terminal BTB domain and a core domain (in
black) with a nuclear localization signal (NLS).
In addition, Lola zf5 and Lola long have tandem zinc finger domains
(Zn). B, diagram of the different 5'-UTR usage by
Lola splice forms. The lola locus has four exons
(I-IV) that code for the common region. The 5'-UTR
utilization for six Lola splice forms are diagrammed. Exons in
black have been confirmed by sequencing, whereas exons in
gray are inferred. In addition, the COOH-terminal sequence
of Lola short (in white) is generated by alternative read
through of exon IV of the common region (10). The insertion site of the
P-element of the lola00642 mutation is indicated by
the triangle.
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Our identification of Lola zf5 as a novel zinc finger-containing
protein within the locus prompted us to survey the region for
additional exons with zinc finger motifs. Consensus zinc finger motifs
include the sequences CXXC, HXXXXC, and
HXXXXH. We searched the lola genomic region for
long open reading frames (ORFs) containing any of these zinc finger
motif consensus sequences. A total of fifteen putative exons containing
zinc finger motifs were identified following this strategy as
summarized in Fig. 4A. From
this analysis we predict that at least 17 splice variants are generated
from the lola locus. Fifteen of these each have a different
zinc finger domain (zf1-zf15), whereas two use exons without zinc
finger motifs. Fig. 4B shows an alignment of the 15 different zinc finger domains within the locus. Two isoforms, Lola zf4
and zf14, have only a single C2H2 zinc finger
domain, whereas the remaining splice variants have tandem
C2HC/C2H2 or
C2HC/C2HC zinc finger domains (Fig.
4B). We propose to name the various novel zinc finger
domain-containing Lola isoforms according to the order of the zinc
finger domain they contain, hence the name Lola zf5. To verify that
these putative isoforms, the majority of which were not predicted by
the genome project, were indeed expressed, we used PCR to amplify
isoform-specific cDNA fragments from the 0- to 21-h yeast
two-hybrid cDNA library based on the assumption that all of the
Lola isoforms share the same common regions as the known isoforms. In
this way the expression of thirteen out of the fifteen predicted zinc
finger-containing exons was confirmed by direct sequencing of such PCR
products (Fig. 4A, indicated in black). In
addition, we searched the Drosophila EST database with the
genomic DNA sequence of each isoform and found further EST support for
expression of six of the 13 exons containing zinc finger motifs. We
also identified an EST clone (GM27815) representing the second Lola
isoform without a zinc finger domain and have named it Lola short-like
(Fig. 4A).

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Fig. 4.
Diagram of the lola genomic
locus and alignment of zinc finger motifs. A, the
lola locus has at least 27 potential exons: four coding for
5'-UTRs, four coding for the Lola common region (I-IV), and
19 alternatively spliced exons coding for the variable COOH-terminal
region of the Lola isoforms. Fifteen of these exons contain zinc finger
domains (ZF1-ZF15). The coding sequence for 17 potential
ORFs of Lola isoforms generated from the locus are diagrammed below.
The existence of the isoforms depicted in black was
supported by Drosophila ESTs from the genome project or by
PCR-amplified isoform-specific cDNA fragments. Lola long and Lola
short have both been previously characterized (10). In addition, a
homolog of Lola zf8 has been identified in D. hydei (11).
B, alignment of the 15 different zinc finger domains from
the zinc finger containing exons. The conserved coordinating cysteines
and histidines are in white typeface outlined in
black.
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The coding regions of most of the Lola isoforms are generated by
splicing the four common exons together with a single exon containing
the different zinc finger domains. However, by sequencing the ESTs and
the PCR amplification products we identified a number of smaller exons
without zinc finger motifs that are utilized by Lola zf1 and zf8 (Fig.
4A). In addition, the locus has four different 5'-UTRs (5'a
through 5'd) that may further amplify the diversity of transcripts
generated from this locus (Fig. 3B). By sequencing ESTs of
the various Lola splice variants, we have obtained evidence that each
of the four 5'-UTRs has been utilized into at least one transcript
(Fig. 3B). Interestingly, we identified two ESTs for Lola
zf5 that used different 5'-UTRs (5'a and 5'c, respectively).
Transcripts with different 5'-UTRs may allow for the fine regulation of
their spatial and temporal expression patterns. Although Lola zf5 is
the only example where this alternative utilization has been confirmed,
it is likely that other isoforms are regulated by the same splicing mechanisms.
Developmental Expression of Lola zf5--
To determine the
expression pattern of Lola zf5, we carried out Northern blot analysis
using mRNA samples from representative developmental stages (Fig.
5). As a probe we used a Lola zf5
isoform-specific cDNA fragment from the COOH-terminal coding region
and 3'-UTR. Lola zf5 mRNA migrates as a single band with an
approximate molecular size of 3.9 kb on 1% agarose-denaturing gels.
Potential differences in size between transcripts using alternative
5'-UTRs would not be resolved on these gels. Lola zf5 mRNA was
abundant in 0- to 2-h embryos, and this level of transcript was
maintained in embryos 2-6 h after egg laying (Fig. 5). However, the
Lola zf5 mRNA level began to decrease in 6- to 12-h embryos and
could not be detected in postembryonic stages except at a low level in
female adults, which may reflect the maternal deposition of mRNA
into eggs. These findings correlated well with the results from
developmental immunoblots using the Lola zf5-specific mAb 7F1 (Fig.
6A). We detected high levels
of Lola zf5 protein in early embryos (0-12 h); however, the protein
level decreased ~12 h after egg laying and could not be detected at
postembryonic stages, including adult females (Fig. 6A). The
lack of Lola zf5 protein in female ovaries suggests that Lola zf5 is
not translated from maternally stored transcripts until after
fertilization. These results indicate that the functional expression of
Lola zf5 is restricted to early embryogenesis.

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Fig. 5.
Developmental Northern blot analysis of Lola
zf5 mRNA. Poly(A)+ mRNA from various stages of
Drosophila development was fractionated on a 1% agarose
denaturing gel, transferred to nylon filter paper, and probed with
random primer-labeled Lola zf5-specific sequences spanning the
COOH-terminal region (upper lanes) or with ribosomal protein
RP49 control probe (lower lanes). A single band of ~3.9 kb
was detected in 0- to 12-h embryos with Lola zf5 probe. Lola zf5
transcripts were not detected in postembryonic stages except at low
levels in female adults.
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Fig. 6.
Western blot analysis of Lola proteins.
A, protein extracts from selected stages of
Drosophila development were fractionated by SDS-PAGE,
immunoblotted, and labeled with the Lola zf5-specific mAb 7F1
(upper lanes) or with tubulin antibody as a control
(lower lanes). The Lola zf5 protein was only detectable
during early embryogenesis and was absent at postembryonic stages.
B, developmental Western blot as in A but probed
with a Lola polyclonal antiserum likely to recognize all the different
Lola isoforms. Multiple Lola isoforms were labeled by the antiserum at
the various developmental stages. C, immunoprecipitation
(ip) of lysates from 0- to 6-h embryos were performed using
Lola zf5 antibody (mAb 7F1) coupled to immunobeads (lane 1 and 2) or with immunobeads only as a control (lane
3) and analyzed by SDS-PAGE and Western blotting using mAb 7F1
(lane 1) and Lola antiserum (lane 2 and
3) for detection. Lola zf5 was detected as a 105-kDa protein
by mAb 7F1 in lane 1. The lower bands
(arrow) are comprised of 7F1 IgG antibody, which was pulled
down in the immunoprecipitation. Rabbit polyclonal Lola antiserum also
recognized the 105-kDa Lola zf5 band but additionally labeled two
protein bands co-immunoprecipitating with Lola zf5. None of the three
bands recognized by the Lola antiserum were detected in the beads only
control lane.
|
|
In previous studies a polyclonal Lola antibody was made to the Lola
common region that would be expected to recognize all the Lola isoforms
(10). In developmental immunoblots using this Lola polyclonal antibody
we detected multiple bands throughout all developmental stages (Fig.
6B). In early embryos (0-6 h) three major bands, including
one migrating at 105 kDa, can be detected on the immunoblots. In 12- to
22-h embryos at least 15 bands can be recognized by the polyclonal Lola
antibody suggesting that most of the Lola isoforms are expressed at
this stage. Some isoforms also appear to be present in later
developmental stages such as third instar larvae as well as adults
(Fig. 6B). To test whether the 105-kDa protein detected by
the Lola antiserum corresponded to Lola zf5 as we would predict, we
performed immunoprecipitation experiments with the mAb 7F1 of protein
extracts from 0- to 6-h embryos. The immunoprecipitations were
fractionated by SDS-PAGE, immunoblotted, and detected with mAb 7F1 and
Lola antiserum, respectively (Fig. 6C). As shown
in Fig. 6C Lola antiserum recognizes three major bands,
including one of 105 kDa that is also labeled by mAb 7F1 and thus is
likely to represent the Lola zf5 protein. Interestingly, the presence
of the two additional bands labeled by the Lola antiserum and not
present in the control lane suggests that Lola zf5 may be involved in
heterodimer formation with other Lola isoforms (Fig. 6C,
lane 2).
The Lethal lola00642 Mutation Is a Dominant Modifier of
the Hypomorphic JIL-1EP(3)3657 Allele--
To further
study whether JIL-1 and Lola zf5 interact in vivo we
explored genetic interactions between mutant alleles of lola and JIL-1 by generating double-mutant individuals containing
both lola00642 and
JIL-1EP(3)3657. The lola00642
allele contains a recessive lethal P element insertion and fails to
complement the lethality of many P element insertion alleles of
lola (40). By PCR amplification of the flanking region of lola00642 followed by direct sequencing, we found
that the insertion site is 438 bp downstream to 5'-UTRc and 54 bp
upstream to 5'-UTRd (Fig. 3B). Because Lola zf5 mRNA
contains either the 5'-UTRa or the 5'-UTRc at the 5'-end of the
transcript, the insertion of a P element within the Lola zf5
transcription unit is likely to disrupt expression of both splicing
alternatives of Lola zf5. The JIL-1EP(3)3657 allele
is a hypomorphic allele that can be maintained in a homozygous stock
for only a few generations due to the low hatch rate and recessive
semi-lethality (6). The hatch rate of JIL-1EP(3)3657
homozygous embryos produced by homozygous parents is as low as 4-7%
when compared with the hatch rate of wild type Oregon-R embryos (Ref. 6
and this study). We generated double mutants by crossing a chromosome
containing the lola00642 allele into a homozygous
JIL-1 hypomorphic EP(3)3657 background. Interestingly, individuals homozygous for
JIL-1EP(3)3657 that also contain a
lola00642 allele can be maintained indefinitely as a
stock. This suggests that heterozygous lola00642 may
function as a dominant suppressor of the
JIL-1EP(3)3657 phenotype. To quantify the extent of
rescue, we compared the numbers of JIL-1EP(3)3657
homozygous progeny with or without lola00642 from a
single cross (Fig. 7A).
Although equal numbers of curly- and straight-winged phenotypic classes
are expected in matings of lola00642/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
males with +/+;
JIL-1EP(3)3657/JIL-1EP(3)3657
females, 2.3 times more flies with straight wings were observed than
curly wings (Fig. 7A). In this cross straight-winged flies carry a lola00642 allele, whereas curly-winged flies
do not. The difference in numbers observed for the two classes was
statistically significant (p < 0.005,
2
test). To exclude the possibility that the apparent rescue phenotype is
due to an enhancer of JIL-1EP(3)3657 phenotype on
the CyO second chromosome balancer, we also set up a control
cross in which the lola00642 chromosome is not
present. In the control cross, we did not observe any statistically
significant evidence (p > 0.1,
2-test)
that the CyO balancer chromosome affects the viability of
JIL-1EP(3)3657 homozygotes (Fig. 7A). The
control crosses were performed by mating +/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
males with +/+;
JIL-1EP(3)3657/JIL-1EP(3)3657
females. To study the strength of rescue of the JIL-1 mutant phenotype by lola00642, we also crossed the
lola00642 mutant chromosome into a heterozygous
JIL-1z2 null background (6). However, we did not
observe any eclosion of z2 homozygotes (data not shown).
These results suggest that either the interaction between
lola00642 and JIL-1 is mild or the
genetic interaction depends on the presence of a minimal level of JIL-1
protein.

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Fig. 7.
Genetic interaction between
lola00642 and
JIL-1EP(3)3657. A, presence of
a heterozygous lola00642 (lola) allele
increases the viability of JIL-1EP(3)3657
(JIL-1) homozygous animals (histograms to the
right). lola00642/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
males were mated with +/+;
JIL-1EP(3)3657/JIL-1EP(3)3657
females. JIL-1EP(3)3657 homozygotes with a
lola00642 allele (histogram in black)
enclosed at a rate 2.3 times greater than
JIL-1EP(3)3657 homozygotes with a wild type
lola allele (histogram in white; normalized to
100%). The difference in numbers observed for the two classes was
statistically significant (p < 0.005, 2
test). Control crosses were performed by mating
+/CyO;JIL-1EP(3)3657/JIL-1EP(3)3657
males with +/+;
JIL-1EP(3)3657/JIL-1EP(3)3657
females. Presence of the CyO balancer alone did not affect
viability of homozygous JIL-1EP(3)3657 animals, as
statistically equivalent (p > 0.1, 2
test) numbers of curly (normalized to 100%) and straight-winged flies
were observed in the F1 progeny (left white and black
histograms). B, rescue of
JIL-1EP(3)3657/JIL-1EP(3)3657
lethality by lola00642 occurs during embryogenesis.
Only 7.2% of embryos from control matings of
JIL-1EP(3)3657/JIL-1EP(3)3657
flies hatched into larvae (white histogram). In contrast,
when lola00642/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
flies were mated, the hatching rate of those embryos not homozygous for
embryonic lethal
lola0062/lola0062 or
CyO/CyO chromosomes increased to 20%
(black histogram). Thus, presence of the heterozygous
lola00642 allele increased the hatch rate of
JIL-1EP(3)3657/JIL-1EP(3)3657
flies 2.8-fold. This difference was statistically significant
(p < 0.005, 2 test). The numbers of
animals counted in all classes are indicated at the bottom
of each histogram. In B the number of embryos hatching from
the total number of lola00642/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
individuals are shown.
|
|
If the observed genetic rescue is a consequence of normalized relative
levels of Lola zf5 and JIL-1, we would expect that some or all of this
rescue occurs during embryogenesis, because Lola zf5 is expressed only
in early embryos (Fig. 6A). We, therefore, quantified
embryonic rescue by determining hatch rates for
JIL-1EP(3)3657/JIL-1EP(3)3657
embryos that were either heterozygous for the
lola00642 allele or homozygous for the wild type
allele. Homozygous
JIL-1EP(3)3657/JIL-1EP(3)3657
embryos produced by homozygous mothers typically hatch at a low (7%)
rate (Fig. 7B). Adjusting for the fact that none of the
embryos with a
lola00642/lola00642 or
CyO/CyO genotype hatch, the hatch rate of
lola00642/CyO;
JIL-1EP(3)3657/JIL-1EP(3)3657
embryos is 20% (Fig. 7B). Thus, homozygous
JIL-1EP(3)3657 embryos that were heterozygous for
lola00642 hatched at a statistically significant
2.8-fold greater rate than embryos that did not carry the
lola mutation (p < 0.005,
2-test). Therefore, the increase of viability observed
for lola and JIL-1 double mutants is at least
partially due to an increase in the frequency with which such
individuals survive embryonic development and hatch.
 |
DISCUSSION |
In this study we provide evidence that the JIL-1 tandem kinase
molecularly interacts with a novel isoform of the lola
locus, Lola zf5. This interaction was first detected in a yeast
two-hybrid screen and subsequently confirmed by pull-down and cross
immunoprecipitation assays. Furthermore, immunocytochemical labeling of
Drosophila embryos shows that Lola zf5 is localized to
nuclei. This localization is compatible with a direct interaction with
JIL-1, because JIL-1 has been shown to be a nuclear kinase expressed
throughout embryogenesis (4). Northern and Western blot analyses show
that the expression of Lola zf5 is developmentally regulated and is
only expressed during early embryogenesis.
An interesting feature of the lola locus is its complex
splicing pattern, and we demonstrate that it contains at least 27 exons. Four of these code for a BTB domain and sequences with a nuclear
localization signal common to all Lola splice forms. In addition,
fifteen of the exons code for sequences with different zinc finger
domains. From this analysis, we predict that a minimum of 17 different
protein products are generated by the lola locus, fifteen of
which contain zinc finger domains and two that do not. By PCR
amplification of cDNAs from 0- to 21-h embryos and sequencing of
ESTs, we have obtained confirming evidence that at least 15 different
Lola polypeptides are likely to be encoded. We were not able to verify
the existence of the two remaining isoforms; however, this could be due
to their being only expressed at developmental stages or in tissues
that we did not examine. We further provide evidence that the number of
transcripts from the locus is enhanced by alternative splicing of four
different 5'-UTRs. The utilization of different 5'-UTRs and exon
shuffling may provide a way to finely regulate stage- and
tissue-specific expression of multiple gene products from the locus
that serve different functional roles. This kind of complex gene
organization has previously been observed at other loci. The most
extreme example may be the Dscam locus that codes for cell
adhesion receptors in the Drosophila nervous system and that potentially can generate more than 38,000 Dscam receptor isoforms (41). Another example related to control of gene
expression is the locus of the trithorax group protein mod(mdg4). This
locus encodes at least 21 different BTB domain-containing protein
isoforms (42). At least one of these isoforms has been shown to
associate with Su(Hw) to exert gypsy insulator function preventing
enhancer-promoter communication (43). Thus, differential splicing may
be a general mechanism for BTB domain proteins to generate functional diversity.
The BTB domain has been shown to promote dimerization and the residues
necessary for this function have been identified for the Bab protein
(16). Comparison between the Lola and Bab BTB domains show that all
these residues are conserved in the Lola BTB domain indicating that it
has the capacity for homodimer formation. Furthermore, our
immunoprecipitation experiments strongly suggest that Lola zf5 forms
heterodimers with other Lola isoforms. Formation of homo- or
heterodimers between Lola isoforms, including Lola zf5 is likely to
lead to different developmental consequences by modifying DNA binding
specificities and/or affinities of the Lola zinc finger isoforms. The
majority of BTB-containing zinc finger proteins described thus far have
been observed to function as transcriptional repressors, and several of
these have been shown to directly bind co-repressor components of
histone deacetylase complexes to their dimerized BTB domains (reviewed
in Ref. 44). Consistent with such a repressive activity it has been
suggested that Lola long may reduce expression of copia
transposable elements in the Drosophila nervous system (11).
However, Cavarec et al. (11) also observed Lola-mediated
positive regulation of copia transcription in the gonads
indicating that Lola isoforms also can act as transcriptional
activators. In support of this notion, they identified a D. hydei isoform of Lola that binds directly to the copia
enhancer and positively regulates transcription in transfected S2
cells. However, this effect was abrogated in a dose-dependent manner when the D. melanogaster Lola long coding sequence was co-transfected. Our
analysis shows that the zinc finger domains of the D. hydei Lola isoform are nearly identical to those of
D. melanogaster Lola zf8, with only a single
conservative substitution of serine to threonine in the second zinc
finger domain. Thus, the expression of alternative Lola isoforms may determine whether Lola acts as an activator or a repressor within different tissues and at specific stages during development.
C2H2 zinc fingers are one of the most common
DNA binding motifs found in eukaryotic transcription factors and are
characterized by a small number of conserved residues that generate the
folded 

domain structure necessary to align the residues that
coordinate binding activity (reviewed in Ref. 45). The identification
of alternatively spliced isoforms of the lola locus
containing different zinc finger arrangements in conjunction with the
ability of Lola isoforms to generate heterodimers suggests that Lola
complexes functioning as transcription factors may recognize a wide
range of potential DNA regulatory sequences. However, it should be
noted that many of the Lola isoforms have a very unusual
arrangement of zinc finger domains that suggest they have the potential
to be involved in protein-protein interactions as well. Although most
zinc fingers are of the C2H2 class, in each of
the Lola isoforms where the zinc finger is present as a tandem array,
the first finger is of the unusual C2HC class. This
atypical zinc finger domain is also found in MYST family histone
acetyltransferases such as MOF, where it has been shown to be essential
for nucleosome interactions (46). Additionally, this domain has been
implicated in binding non-histone proteins (47), RNA (48-50), and DNA
(51, 52).
Our genetic experiments suggest that a reduction of protein levels
resulting from mutation in the lola locus can act as a dominant modifier of a hypomorphic JIL-1 allele leading to
an increase in embryonic viability. However, in these experiments the P
element insertion into the lola locus is likely to perturb transcription of many of the Lola isoforms. Thus, we do not know which
of the Lola isoforms are responsible for the genetic interaction or
whether JIL-1 acts upstream or downstream. Thus, several scenarios for
a functional interaction between Lola proteins and JIL-1 that enhances
viability can be envisioned. On one hand, JIL-1 may act as a
derepressor to counteract a potential repressive function of Lola zf5
or other Lola isoforms on gene expression. Derepression of the gene
products from these loci may lead to enhanced viability. On the other
hand, Lola zf5 or other Lola isoforms may normally act to down-regulate
JIL-1 kinase activity by physically interacting with JIL-1.
Consequently, the decrease in JIL-1 kinase activity observed in a
hypomorphic mutant background may be alleviated by the reduction of its
negative regulator in the lola mutant. In a third scenario,
the interaction of JIL-1 with Lola zf5 may indeed enhance transcription
at some genes, but the loss in the lola mutant of other Lola
isoforms that normally function to down-regulate gene expression in
other contexts may counterbalance the reduced JIL-1 activity in the
JIL-1 hypomorph. Thus, the interaction between JIL-1 and the
lola locus may be highly complex and promises to provide new
experimental avenues into exploring the mechanisms of modification of
chromatin and/or the regulation of gene expression during early embryogenesis.
We thank Dr. E. Giniger for the gift of the
Lola polyclonal antiserum, Dr. D. Zhao for generously providing the
SW59 clone, and the Iowa State University Cell and Hybridoma Facility
for assistance in antibody production. We thank Dr. L. Ambrosio and members of the laboratory for discussion, advice, and critical reading
of the manuscript. We also wish to thank V. Lephart for maintenance of fly stocks.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M213269200
The abbreviations used are:
MSL, male-specific
lethal;
mAb, monoclonal antibody;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
KDI, kinase
domain I;
EST, expressed sequence tag;
ORF, open reading frame;
UTR, untranslated region;
TRITC, tetramethylrhodamine
isothiocyanate.
1.
|
Varga-Weisz, P. D.,
and Becker, P. B.
(1998)
Curr. Opin. Cell Biol.
10,
346-353[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Wolffe, A. P.,
and Hayes, J. J.
(1999)
Nucleic Acids Res.
27,
711-720[Abstract/Free Full Text]
|
3.
|
Johansen, K. M.,
Johansen, J.,
Jin, Y.,
Walker, D. L.,
Wang, D.,
and Wang, Y.
(1999)
Crit. Rev. Eukaryotic Gene Expr.
9,
267-277[Medline]
[Order article via Infotrieve]
|
4.
|
Jin, Y.,
Wang, Y.,
Walker, D. L.,
Dong, H.,
Conley, C.,
Johansen, J.,
and Johansen, K. M.
(1999)
Mol. Cell
4,
129-135[Medline]
[Order article via Infotrieve]
|
5.
|
Jin, Y.,
Wang, Y.,
Johansen, J.,
and Johansen, K. M.
(2000)
J. Cell Biol.
149,
1005-1010[Abstract/Free Full Text]
|
6.
|
Wang, Y.,
Zhang, W.,
Jin, Y.,
Johansen, J.,
and Johansen, K. M.
(2001)
Cell
105,
433-443[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Meller, V. H.,
and Kuroda, M. I.
(2002)
Adv. Genet.
46,
1-24[Medline]
[Order article via Infotrieve]
|
8.
|
Smith, E. R.,
Pannuti, A.,
Gu, W.,
Seurnagel, A.,
Cook, R. G.,
Allis, C. D.,
and Lucchesi, J. C.
(2000)
Mol. Cell. Biol.
20,
312-318[Abstract/Free Full Text]
|
9.
|
Seeger, M.,
Tear, G.,
Ferres-Marco, D.,
and Goodman, C. S.
(1993)
Neuron
10,
409-426[Medline]
[Order article via Infotrieve]
|
10.
|
Giniger, E.,
Tietje, K.,
Jan, L. Y.,
and Jan, Y. N.
(1994)
Development
120,
1385-1398[Abstract/Free Full Text]
|
11.
|
Cavarec, L.,
Jensen, S.,
Casella, J.-F.,
Cristescu, S. A.,
and Heidmann, T.
(1997)
Mol. Cell. Biol.
17,
482-494[Abstract]
|
12.
|
Crowner, D.,
Madden, K.,
Goeke, S.,
and Giniger, E.
(2002)
Development
129,
1317-1325[Medline]
[Order article via Infotrieve]
|
13.
|
Godt, D.,
Couderc, J. L.,
Cramton, S. E.,
and Laski, F. A.
(1993)
Development
119,
799-812[Abstract/Free Full Text]
|
14.
|
Zollman, S.,
Godt, D.,
Prive, G. G.,
Couderc, J. L.,
and Laski, F. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10717-10721[Abstract/Free Full Text]
|
15.
|
Bardwell, V. J.,
and Treisman, R.
(1994)
Genes Dev.
8,
1664-1677[Abstract]
|
16.
|
Chen, W.,
Zollman, S.,
Couderc, J.-L.,
and Laski, F. A.
(1995)
Mol. Cell. Biol.
15,
3424-3429[Abstract]
|
17.
|
Ahmad, K. F.,
Engel, C. K.,
and Privé, G. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12123-12128[Abstract/Free Full Text]
|
18.
|
Kobayashi, A.,
Yamagiwa, H.,
Hoshino, H.,
Muto, A.,
Sato, K.,
Morita, M.,
Hayashi, N.,
Yamamoto, M.,
and Igarashi, K.
(2000)
Mol. Cell. Biol.
20,
1733-1746[Abstract/Free Full Text]
|
19.
|
Kanezaki, R.,
Toki, T.,
Yokoyama, M.,
Yomogida, K.,
Sugiyama, K.,
Yamamoto, M.,
Igarashi, K.,
and Ito, E.
(2001)
J. Biol. Chem.
276,
7278-7284[Abstract/Free Full Text]
|
20.
|
Albagli, O.,
Dhordain, P.,
Deeweindt, C.,
Lecocq, G.,
and Leprince, D.
(1995)
Cell Growth Differ.
6,
1193-1198[Abstract]
|
21.
|
Huynh, K. D.,
and Bardwell, V. J.
(1998)
Oncogene
17,
2473-2484[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Lin, R. J.,
Nagy, L.,
Inoue, S.,
Shao, W.,
Miller, W. H., Jr.,
and Evans, R. M.
(1998)
Nature
391,
811-814[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Wong, C.-W.,
and Privalsky, M. L.
(1998)
J. Biol. Chem.
273,
27695-27702[Abstract/Free Full Text]
|
24.
|
Melnick, A.,
Carlile, G.,
Ahmad, K. F.,
Kiang, C.-L.,
Corcoran, C.,
Bardwell, V.,
Prive, G. G.,
and Licht, J. D.
(2002)
Mol. Cell. Biol.
22,
1804-1818[Abstract/Free Full Text]
|
25.
|
Heinzel, T.,
Lavinsky, R. M.,
Mullen, T.-M.,
Söderström, M.,
Laherty, C. D.,
Torchia, J.,
Yang, W.-M.,
Brard, G.,
Ngo, S. D.,
Davie, J. R.,
Seto, E.,
Eisenman, R. N.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1997)
Nature
387,
43-48[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Alland, L.,
Muhle, R.,
Hou, H., Jr.,
Potes, J.,
Chin, L.,
Schreiber-Agus, N.,
and DePinho, R. A.
(1997)
Nature
387,
49-55[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Wolffe, A. P.
(1996)
Science
272,
408-411[Abstract]
|
28.
|
Madden, K.,
Crowner, D.,
and Giniger, E.
(1999)
Dev. Biol.
213,
301-313[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Roberts, D. B.
(1986)
Drosophila: A Practical Approach
, IRL Press, Oxford, UK
|
30.
|
Lindsley, D. L.,
and Zimm, G. G.
(1992)
The Genome of Drosophila melanogaster
, Academic Press, New York
|
31.
|
Sambrook, J.,
and Russell, D. W.
(2001)
Molecular Cloning: A Laboratory Manual
, 3rd Ed.
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
|
32.
|
Adams, M. D.,
Celniker, S. E.,
Holt, R. A.,
Evans, C. A.,
Gocayne, J. D.,
Amanatides, P. G.,
Scherer, S. E.,
Li, P. W.,
Hoskins, R. A.,
Galle, R. F.,
George, R. A.,
Lewis, S. E.,
Richards, S.,
Ashburner, M.,
Henderson, S. N.,
Sutton, G. G.,
Wortman, J., R.,
Yandell, M. D.,
Zhang, Q.,
Chen, L. X.,
Brandon, R. C.,
Rogers, Y. H.,
Blazej, R. G.,
Champe, M.,
Pfeiffer, B. D.,
Wan, K. H.,
Doyle, C.,
Baxter, E. G.,
Helt, G.,
Nelson, C. R.,
Gabor, G. L.,
Abril, J. F.,
Agbayani, A.,
An, H. J.,
Andrews-Pfannkoch, C.,
Baldwin, D.,
Ballew, R. M.,
Basu, A.,
Baxendale, J.,
Bayraktaroglu, L.,
Beasley, E. M.,
Beeson, K. Y.,
Benos, P. V.,
Berman, B. P.,
Bhandari, D.,
Bolshakov, S.,
Borkova, D.,
Botchan, M. R.,
Bouck, J.,
Brokstein, P.,
Brottier, P.,
Burtis, K. C.,
Busam, D. A.,
Butler, H.,
Cadieu, E.,
Center, A.,
Chandra, I.,
Cherry, J. M.,
Cawley, S.,
Dahlke, C.,
Davenport, L. B.,
Davies, P.,
de Pablos, B.,
Delcher, A.,
Deng, Z.,
Mays, A. D.,
Dew, I.,
Dietz, S. M.,
Dodson, K.,
Doup, L. E.,
Downes, M.,
Dugan-Rocha, S.,
Dunkov, B. C.,
Dunn, P.,
Durbin, K. J.,
Evangelista, C. C.,
Ferraz, C.,
Ferriera, S.,
Fleischmann, W.,
Fosler, C.,
Gabrielian, A. E.,
Garg, N. S.,
Gelbart, W. M.,
Glasser, K.,
Glodek, A.,
Gong, F.,
Gorrell, J. H.,
Gu, Z.,
Guan, P.,
Harris, M.,
Harris, N. L.,
Harvey, D.,
Heiman, T. J.,
Hernandez, J. R.,
Houck, J.,
Hostin, D.,
Houston, K. A.,
Howland, T. J.,
Wei, M. H.,
Ibegwam, C.,
Jalali, M.,
Kalush, F.,
Karpen, G. H.,
Ke, Z.,
Kennison, J. A.,
Ketchum, K. A.,
Kimmel, B. E.,
Kodira, C. D.,
Kraft, C.,
Kravitz, S.,
Kulp, D.,
Lai, Z.,
Lasko, P.,
Lei, Y.,
Levitsky, A. A.,
Li, J.,
Li, Z.,
Liang, Y.,
Lin, X.,
Liu, X.,
Mattei, B.,
McIntosh, T. C.,
McLeod, M. P.,
McPherson, D.,
Merkulov, G.,
Milshina, N. V.,
Mobarry, C.,
Morris, J.,
Moshrefi, A.,
Mount, S. M.,
Moy, M.,
Murphy, B.,
Murphy, L.,
Muzny, D. M.,
Nelson, D. L.,
Nelson, D. R.,
Nelson, K. A.,
Nixon, K.,
Nusskern, D. R.,
Pacleb, J. M.,
Palazzolo, M.,
Pittman, G. S.,
Pan, S.,
Pollard, J.,
Puri, V.,
Reese, M. G.,
Reinert, K.,
Remington, K.,
Saunders, R. D.,
Scheeler, F.,
Shen, H.,
Shue, B. C.,
Siden-Kiamos, I.,
Simpson, M.,
Skupski, M. P.,
Smith, T.,
Spier, E.,
Spradling, A. C.,
Stapleton, M.,
Strong, R.,
Sun, E.,
Svirskas, R.,
Tector, C.,
Turner, R.,
Venter, E.,
Wang, A. H.,
Wang, X.,
Wang, Z. Y.,
Wassarman, D. A.,
Weinstock, G. M.,
Weissenbach, J.,
Williams, S. M.,
Woodage, T.,
Worley, K. C.,
Wu, D.,
Yang, S.,
Yao, Q. A.,
Ye, J.,
Yeh, R. F.,
Zaveri, J. S.,
Zhan, M.,
Zhang, G.,
Zhao, Q.,
Zheng, L.,
Zheng, X. H.,
Zhong, F. N.,
Zhong, W.,
Zhou, X.,
Zhu, S.,
Zhu, X.,
Smith, H. O.,
Gibbs, R. A.,
Myers, E. W.,
Rubin, G. M.,
and Venter, J. C.
(2000)
Science
287,
2185-2195[Abstract/Free Full Text]
|
33.
|
Rubin, G. M.,
Hong, L.,
Brokstein, P.,
Evans-Holm, M.,
Frise, E.,
Stapleton, M.,
and Harvey, D. A.
(2000)
Science
287,
2222-2224[Abstract/Free Full Text]
|
34.
|
Harlow, E.,
and Lane, E.
(1988)
Antibodies: A Laboratory Manual
, Cold Spring Harbor Press, Cold Spring Harbor, NY
|
35.
|
Mitchison, T.,
and Sedat, J.
(1983)
Dev. Biol.
99,
261-264[Medline]
[Order article via Infotrieve]
|
36.
|
Ruhf, M.-L.,
Braun, A.,
Papoulas, O.,
Tamkun, J. W.,
Randsholt, N.,
and Meister, M.
(2001)
Development
128,
1429-1441[Abstract/Free Full Text]
|
37.
|
Usuka, J.,
and Brendel, V.
(2000)
J. Mol. Biol.
297,
1075-1085[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Tatusova, T. A.,
and Madden, T. L.
(1999)
FEMS Microbiol. Lett.
174,
247-250[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Higgins, D. G.,
Thompson, J. D.,
and Gibson, T. J.
(1996)
Methods Enzymol.
266,
383-402[Medline]
[Order article via Infotrieve]
|
40.
|
Spradling, A. C.,
Stern, D.,
Beaton, A.,
Rhem, E. J.,
Laverty, T.,
Mozden, N.,
Misra, S.,
and Rubin, G. M.
(1999)
Genetics
153,
135-177[Abstract/Free Full Text]
|
41.
|
Schmucker, D.,
Clemens, J. C.,
Shu, H.,
Worby, C. A.,
Xiao, J.,
Muda, M.,
Dixon, J. E.,
and Zipursky, S. L.
(2000)
Cell
101,
671-684[Medline]
[Order article via Infotrieve]
|
42.
|
Büchner, K.,
Roth, P.,
Schotta, G.,
Krauss, V.,
Saumweber, H.,
Reuter, G.,
and Dorn, R.
(2000)
Genetics
155,
141-157[Abstract/Free Full Text]
|
43.
|
Ghosh, D.,
Gerasimova, T. I.,
and Corces, V. G.
(2001)
EMBO J.
20,
2518-2527[Abstract/Free Full Text]
|
44.
|
Deltour, S.,
Guerardel, C.,
and Leprince, D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14831-14836[Abstract/Free Full Text]
|
45.
|
Wolfe, S. A.,
Nekludova, L.,
and Pabo, C. O.
(2000)
Annu. Rev. Biophys. Biomol. Struct.
29,
183-212[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Akhtar, A.,
and Becker, P. B.
(2001)
EMBO Rep.
2,
113-118[Abstract/Free Full Text]
|
47.
|
Burke, T. W.,
Cook, J. G.,
Asano, M.,
and Nevins, J. R.
(2001)
J. Biol. Chem.
276,
15397-15408[Abstract/Free Full Text]
|
48.
|
Arrizabalaga, G.,
and Lehmann, R.
(1999)
Genetics
153,
1825-1838[Abstract/Free Full Text]
|
49.
|
Gorelick, R. J.,
Fu, W.,
Gagliardi, T. D.,
Bosche, W. J.,
Rein, A.,
Henderson, L. E.,
and Arthur, L. O.
(1999)
J. Virol.
73,
8185-8195[Abstract/Free Full Text]
|
50.
|
Urbaneja, M. A.,
Kane, B. P.,
Johnson, D. G.,
Gorelick, R. J.,
Henderson, L. E.,
and Casas-Finet, J. R.
(1999)
J. Mol. Biol.
287,
59-75[CrossRef][Medline]
[Order article via Infotrieve]
|
51.
|
Kim, J. G.,
Armstrong, R. C.,
v Agoston, D.,
Robinsky, A.,
Wiese, C.,
Nagle, J.,
and Hudson, L. D.
(1997)
J. Neurosci. Res.
50,
272-290[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Lee, C. C.,
Beall, E. L.,
and Rio, D. C.
(1998)
EMBO J.
17,
4166-4174[Abstract/Free Full Text]
|