kinase suppressor of ras
Two genes in Drosophila have been identified and characterized whose products are required for activated RAS to signal with normal efficiency, but they do not appear to effect signaling by activated RAF. One encodes the beta subunit of type I geranylgeranyl transferase, a prenylation enzyme essential for targeting RAS to the plasma membrane. The other encodes a protein kinase that has been named Kinase suppressor of ras. KSR functions in multiple receptor tyrosine kinase pathways. Mammalian homologs of KSR have been identified that, together with the Drosophila gene, define a novel class of kinases. These results suggest that KSR is a general and evolutionarily conserved component of the RAS signaling pathway that acts between RAS and RAF (Therrien, 1995).
kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).
Complementation test results reveal that SK2-4 is allelic to Src42A, the closest Drosophila homolog of the Src kinase family. Intriguingly, genetic data (suppression of sE-KDN, enhancement of sev-Ras1V12, and suppression of RafHM7) suggest an inhibitory role for Src42A in Ras1 signaling, which is the opposite of the described function of Src-like kinases in vertebrates. During the course of this work, Src42A was also identified by another laboratory as being a dominant suppressor of a hypomorphic allele of Raf (RafC110). Genetic and molecular characterization of different Src42A alleles clearly supports an inhibitory function for Src42A in different RTK-dependent signaling pathways. Therefore, the further elucidation of the molecular function of Src42A in Drosophila may unveil a novel mechanism of action for this family of nonreceptor tyrosine kinases (Therrien, 2000).
Since alleles of EK2-9 failed to complement two independent P elements recently shown to disrupt the RRM-motif protein locus split ends (spen), it is concluded that EK2-9 is allelic to spen. This gene encodes at least three large (~5500 amino acids) and closely related protein isoforms. The Spen proteins contain three RRMs at the N terminus and a conserved region of unknown function at the C terminus. The presence of three RRMs suggests that the Spen proteins mediate their effect via a RNA binding-dependent mechanism such as RNA processing or transport. Interestingly, mutant alleles of spen have been isolated in several independent genetic screens in Drosophila. They were initially recovered in a screen for mutations affecting peripheral nervous system development. Subsequently, they were isolated in two related screens that, like the KDN screen, were designed to identify novel components of the Ras1 pathway. Mutations in spen were isolated as dominant enhancers of a Raf-induced [E(Raf) 2A complementation group] and a Yan-induced rough eye phenotype. The fact that three separate screens targeting Ras1 signaling (KSR-, Raf-, and Yan-dependent) recovered mutant alleles of spen suggests that this locus is relevant for Ras1-mediated signal transduction. Examination of the genetic interactions, however, indicates that the role of spen in Ras1 signaling may not be straightforward. For example, the ability of the E(Raf)2A/spen alleles to dominantly enhance an activated Raf-dependent phenotype suggests that spen is a negative regulator of the pathway. However, alleles of spen also enhance RafHM7 lethality at 18°, and homozygous mutant clones in the eye are often missing R7 and outer photoreceptor cells, although extra photoreceptor cells are occasionally found. These results are more consistent with a positive role for spen during Ras1 signaling and would agree with the fact that spen mutations have been recovered as enhancers of sE-KDN and gmr-yanact. Alleles of spen were also recovered as enhancers of a loss-of-function phenotype for the Hox gene Deformed (Dfd). In that context, the genetic analysis of spen suggests that it functions in parallel to Dfd for the specification of head cuticular structures. Finally, mutations in spen have been identified as dominant enhancers of an E2F/Dp-induced rough eye phenotype and as dominant suppressors of p21CIP1-induced phenotypes. The fact that S-phase entry is stimulated by the overexpression of E2F and Dp proteins in the eye but inhibited by p21CIP1 overexpression suggests that spen may be involved in the negative regulation of cell cycle progression (Therrien, 2000).
Currently it is unknown how spen activity links Ras1-dependent cell differentiation, Hox-dependent segment specification, and E2F-dependent cell cycle control. Nonetheless, their common requirement for spen function suggests that they are interconnected. In agreement with this idea, other loci have been found in common in the screens mentioned above, as well as in other related screens. One of these loci corresponds to the kismet (kis) gene. In addition to the EK2-4/kis alleles identified in this KDN screen, mutations in kis were recovered as dominant enhancers in the Dfd screen and as dominant suppressors in a Polycomb (Pc) loss-of-function screen. Alleles of kis have also been identified as dominant suppressors of the synthetic lethality generated by the coexpression of activated Sevenless (SevS11) and Ras1V12 (Therrien, 2000).
The ability of kis mutations to suppress the homeotic transformations observed in a Pc mutant background and to suppress the expression of the Hox gene Sex combs reduced suggests that kis is a member of the trithorax group (trxG) of genes. In Drosophila, trxG genes are essential for Hox gene expression, where they appear to counteract the repressive effect of the Polycomb group (PcG) of genes on Hox gene expression. Interestingly, a number of trxG proteins are similar to components of the large (2 MD) ATPase-dependent SWI/SNF complex that facilitates the access of transcription factors to their DNA binding sites by destabilizing the nucleosomes. It is thus possible that trxG proteins control Hox gene expression by directly altering the chromatin at specific Hox loci. Strikingly, sequence analysis of the Kis proteins reveals strong homologies with the CHD proteins, which represent a novel class of putative ATPase subunits of chromatin remodeling complexes, and with Brahma, a trxG protein that is the closest fly homolog to the yeast SWI2/SNF2 ATPase subunit. These observations strongly suggest that the Kis proteins are also involved in chromatin remodeling. Interestingly, Brahma and Kis proteins do not appear to coexist in a common complex. This raises the possibility that Kis is part of a different chromatin remodeling complex that might regulate the transcription of a distinct group of genes. Consistent with this hypothesis, brahma mutations have not been isolated in the Ras1 pathway-dependent screens mentioned above. On the basis of the presumed function for Kis and the observation that kis loss-of-function mutations suppress sev-Ras1V12, and enhance sE-KDN and RafHM7 rough eye phenotypes, it is tempting to speculate that Kis is part of a complex that, in response to Ras1-dependent signals, directly alters the transcription of a specific group of genes required for Ras1-dependent cell differentiation during Drosophila eye development (Therrien, 2000).
Incubation of cells with dsRAS1 RNA prevents MEK and MAPK activation. dsRAS1 RNA specifically decreases the levels of RAS1, but does not affect the levels of endogenous RAF, MEK, and MAPK. Specific removal of the three kinases of the module also impairs signal transduction. However, elimination of MAPK in S2 cells leads to an increase in pMEK levels, which suggests the presence of a MAPK-dependent negative-feedback mechanism. Interestingly, ablation of endogenous KSR also precludes activation of MEK and MAPK by RASV12 and similar results are obtained when the pathway is activated using an activated form of the Sevenless receptor tyrosine kinase, which indicates that KSR plays a critical role as well in situations where RAS has not been overexpressed. Therefore, these results show that KSR is a bona fide component of the MAPK pathway. In addition, given that dsKSR RNA inhibits MEK activation, it implies that KSR is required at a step upstream of MEK. This is consistent KSR's ability to promote MEK phosphorylation (Roy, 2002).
CNK, a RAF-binding multidomain protein required for RAS signaling
kinase suppressor of ras (ksr) is required for efficient signal transmission within the Ras/MapK cascade. A screen for mutations that modify a ksr-dependent phenotype has identified a novel gene, connector enhancer of ksr (cnk), which functions either upstream or in parallel to Raf in the Ras pathway. cnk encodes a protein of 1557 amino acids containing several protein-protein interaction domains, suggesting that Cnk brings different signaling molecules together. Searches of protein and translated nucleotide databases reveal a multidomain organization for the Cnk protein. These domains include a sterile alpha motif (SAM) domain, a PSD-95/DLG-1/ZO-1 (PDZ) domain, and a pleckstrin homology (PH) domain. In addition, protein sequence analysis has identified two proline-rich stretches containing SH3 domain consensus binding sites and several potential tyrosine phosphorylation sites corresponding to different SH2 domain consensus binding sites. This structural organization is reminiscent of multiadaptor proteins and suggests that the function of Cnk is to bring together different molecules. A search of the expressed sequence tags (EST) database identified several sequences encoding SAM, PDZ, or PH domains similar to those in CNK. Full-length cDNA clones were isolated corresponding to a few of these ESTs and their sequences were determined. One of them encodes a protein of 751 amino acids that, although smaller than CNK, has a similar modular arrangement: a SAM domain at the extreme N terminus, followed by a PDZ domain and then a PH domain. A putative C. elegans ORF contains the same organization of protein modules. A closer inspection of sequence similarities between these three proteins identified another conserved region of about 80 amino acids between the SAM and PDZ domains that might represent a novel protein module. This putative domain was named "CRIC" for conserved region in CNK (Therrien, 1998).
To determine whether cnk has a role during Ras signaling, the ability of cnk to modify the rough eye phenotype caused by overexpression of Ras1V12 (constitutively active Ras), sevS11 (activated Sevenless RTK, and RafTor4021 (activated RAF) was tested. cnk dominantly suppresses both Ras1V12 and sevS11 rough eye phenotypes but is unable to modify the RafTor4021 rough eye phenotype. Analysis of apical sections of adult fly retinae support this conclusion, and multiple cnk alleles have the same effect. The ability of cnk to enhance KDN (dominant negative Kinase suppressor of Ras) and to suppress both Ras1V12 and sevS11 phenotypes suggests that the EK2-3 locus encodes a positive component of RAS signaling. In addition, the finding that cnk does not modify the RafTor4021 phenotype suggests that cnk is required upstream or in parallel to Raf. The finding that cnk loss-of-function alleles modify sensitized genetic backgrounds like those produced by KDN or sev-Ras1V12 suggests that this gene is normally required for photoreceptor differentiation. This conclusion is supported by examination of the mild rough eyes of a hypomorphic allele. Analysis of apical eye sections of hypomorphic allele homozygotes reveals the absence of R7 photoreceptors in more than 80% of the ommatidia, with roughly 15% of the ommatidia also missing an outer photoreceptor. This phenotype is similar to that observed for hypomorphic alleles of several components of the Ras pathway. Because R7 and outer photoreceptor cell differentiation depend on Sevenless and Epidermal growth factor, respectively, this observation suggests that cnk functions downstream of both receptors (Therrien, 1998).
In Drosophila, components of the RAS pathway are generally required for cell proliferation and/or survival. To test whether cnk is similarly required, large clones of cnk homozygous mutant tissue were generated in the eye. cnk appears to be required for cell proliferation and/or survival, since mutation of this gene prevents normal eye development in a similar manner as is observed when loss-of-function mutations in Ras1 or ksr are tested. Evidence is found indicating that cnk is required for RAS-dependent signaling pathways outside the eye. For example, like other components of this pathway, cnk loss-of-function alleles are synthetic lethal with a hypomorphic allele of Raf, RafHM7. Induction of cnk mutant clones in the wing prevents wing vein formation. Wing vein formation is controlled by Egfr, indicating a role for cnk in other RAS-dependent pathways (Therrien, 1998).
To identify a putative site of action for Cnk, its localization within the cell was determined. The protein is mainly confined to the apical portions of cells where adherens junctions are found. In addition, analysis of several focal planes reveals a significant membrane localization throughout the cells, but no cytoplasmic or nuclear staining. The apical localization of Cnk is similar to that of Sevenless and the two adaptor molecules Drk and Dos. In cultured cells, Cnk is found in the cytoplasm, but, interestingly, it also accumulates in the membrane at cell-cell contact points. Since Cnk and Sevenless appear to colocalize in the eye disc and the Cnk amino acid sequence contains several SH2 domain consensus binding sites, it was asked whether Cnk could be tyrosine phosphorylated in a Sevenless-dependent manner. Constitutively active Sevenless induces tyrosine phosphorylation of Cnk. Cnk is also a target for Egf-dependent tyrosine phosphorylation in cultured cells. These results do not distinguish whether these RTKs directly phosphorylate Cnk or induce another tyrosine kinase, which in turn phosphorylates Cnk (Therrien, 1998).
To determine if an increase in wild-type Cnk level could transform cone cells into R7 cells, the effect of overexpressing wild-type Cnk was examined in the developing eye. Adult flies overexpressing Cnk have a mild rough eye phenotype. Apical sections of these eyes reveal missing R7 and outer photoreceptor cells, indicating that when overexpressed in the eye, wild-type CNK has a mild dominant-negative effect. This effect might be explained by the titration of one or more CNK-interacting proteins that are normally required for endogenous Ras signaling. The absence of extra R7 cells indicates that overexpression of Cnk is not sufficient to activate the Ras pathway. Cnk overexpression massively enhances an activated Ras1 phenotype. This result indicates that CNK cooperates with activated RAS1 and suggests that CNK function is RAS dependent. Cnk is shown to physically interact with the C-terminal kinase domain of Raf and appears to localize to cell-cell contact regions. Together, these findings suggest that Cnk is a novel component of a Ras-dependent signaling pathway that regulates Raf function and/or targets Raf to a specific subcellular compartment upon Ras activation (Therrien, 1998).
Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screen was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. The KSR kinase domain functions as a dominant-negative molecule when separated from the noncatalytic N-terminal domain (Therrien, 1996).
Overexpression of a KSR dominant-negative (KDN) protein in a subset of cells of the developing eye under the control of the sevenless enhancer/heat shock promoter (sE-KDN) blocks RAS1-dependent photoreceptor cell differentiation. In sE-KDN ommatidia, the R7 photoreceptor cell and one or two of the other photoreceptor cells are missing. Thus, most ommatidia have five to seven photoreceptor cells instead of the normal eight, resulting in a visible rough eye phenotype, which is clearly distinguishable under a dissecting microscope (Therrien, 1996, 1998, 2000). The rough eye phenotype generated in this manner is sensitive to the copy number of transgenes; that is, flies carrying two copies of the transgene have more severely disrupted eyes than flies carrying only one copy. The dose sensitivity of this phenotype has provided a good background for a genetic screen designed to identify loss-of-function mutations that dominantly modify this phenotype by disruption of one of two copies of the wild-type gene (Therrien, 1998, 2000). In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were also recovered: one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).
One of the misexpression interactors, MESR2, is an insertion upstream of the Akap200 locus. DAKAP200 refers to Drosophila A kinase anchor protein of molecular weight 200 kd, and binds the regulatory II (rII) subunit of cyclic AMP-dependent protein kinase (PKA). The Akap200 gene produces two different transcripts, one that contains the binding site for RII and one where the exon encoding for the RII binding site is spliced out to generate a protein that does not interact directly with PKA. Both isoforms of AKAP200 are expressed at relatively similar levels throughout development as well as in adult heads (Huang, 2000).
PKA is the principal mediator of signals that activate adenylate cyclase. cAMP signals are often targeted to effectors that accumulate to discrete intracellular locations. This targeting is due to a nonuniform distribution of PKA molecules within cells. In Drosophila, PKA has been implicated in normal developmental events in all imaginal tissues through the Hedgehog signaling pathway and is involved in signaling pathways that generate cell polarity: this requires that Hh be localized to distinct intracellular locations. Subcellular localization of PKA occurs through association with AKAPs. AKAPs are a functionally related family of proteins, defined by their ability to associate with PKA. Each AKAP contains a unique targeting domain that directs the complex to a defined intracellular location where PKA is placed proximal to both a signal generator (adenylate cyclase) as well as to potential PKA effector molecules. Coordinate binding of specific combinations of enzymes can allow such complexes to respond to distinct second messenger-mediated signals (Huang, 2000).
Studies in mammalian cells have suggested that PKA signaling via Rap1, another small molecular weight GTP-binding protein, antagonizes RAS1 signaling by competing for RAS pathway components such as B-Raf and MAPK. However, more recent studies suggest no genetic interaction between Drosophila Rap1 and RAS1. In Drosophila, overexpression of Rap1 in a heterozygous RAS1 mutant background has no effect on photoreceptor determination, suggesting no interaction between the two gene functions. A heterozygous Rap1 mutation does not reduce the number of R7 cells in a sev-RAS1V12 rough eye, also suggesting that the two pathways do not interact. Although there is no direct evidence linking PKA activation to MAPK activation via Rap1, there may be a still unknown pathway by which these molecules can signal (Huang, 2000).
The screen isolated Akap200 as a misexpression enhancer of KDN and suppressor of RAS1V12. This suggests that overexpression of this AKAP decreases signaling through RAS1. Overexpression of an AKAP might cause mislocalization of PKA molecules to the plasma membrane. This could activate a signaling pathway that normally is not utilized in this cell or at this time in development. If PKA and Rap1 are involved in RAS signaling, why were they not uncovered in previous loss-of-function screens? One possibility is that mutations in either gene may not be dose sensitive and therefore be unable to dominantly modify a rough eye phenotype. Another is the possibility that overexpression of an AKAP causes abnormal targets of PKA to become activated. Whether PKA signals through Rap1 remains unclear; however, the reported effects of attenuating RAS1/MAPK signaling are supported by this study. The enhancement of the KDN rough eye phenotype could be due to the additive effects of inefficient signaling due to KDN as well as the attenuation of MAPK by mislocalized PKA. In the activated RAS1V12 background, the attenuating effects of activated PKA due to mislocalization to the plasma membrane might reduce the amount of signaling through the pathway to suppress the RAS1-dependent rough eye phenotype (Huang, 2000).
Overexpression of msn in an sE-KDN background enhances the rough eye phenotype. msn encodes the Drosophila homolog of Nck interacting kinase (NIK), a member of the mammalian SPS1 subfamily of the STE20 kinase family. msn is an essential gene involved in dorsal closure during embryogenesis in Drosophila and mutant clones result in misshapen rhabdomeres (due to defects in polarity, malformed and missing photoreceptors) in the adult eye. STE20, the founding member of the family in yeast, acts in the pheromone signaling pathway and activates the yeast MAPK module. However, the upstream activators of the STE20 pathway are not known. msn, the STE20 homolog in Drosophila, acts upstream of the c-Jun amino-terminal kinase (JNK) MAPK cascade required for dorsal closure and has recently been implicated to act downstream of the Frizzled receptor in the epithelial planar polarity pathway. Published results have suggested that msn, when overexpressed in the eye in an otherwise wild-type background, can generate a rough eye. However, in the experiments described here, no effect on eye morphology was found using sE-GAL4 to drive either UAS-msn or EP(3)0609 and EP(3)0549, the two EP lines upstream of the msn gene. Drosophila Jun has been implicated as a downstream target of both the JNK MAPK and RAS1/MAPK signal transduction pathways by overexpression analysis of dominant-negative mutations; however, no other components of either pathway are shared. If the JNK pathway can partially compensate for the RAS1 pathway in eye development, one would expect that msn overexpression would suppress sE-KDN. The overexpression results from this screen suggest that as a misexpression suppressor of RAS1, msn decreases signaling in the pathway. msn may independently inhibit neuronal cell fate, although there is no previous evidence for this. It is possible that the JNK signaling pathway may compete with the RAS pathway for common components. Alternatively, this interaction may be tissue specific and only uncovered in the eye with overexpression. There is also the possibility that misexpression of msn causes promiscuous signaling through an independent pathway that also affects eye morphology. Although no phenotype is seen when msn is overexpressed in the eye, this situation may sensitize the eye and nonspecifically enhance the sE-KDN phenotype (Huang, 2000 and references therein).
Another misexpression interactor, MESR8, corresponds to two independent insertions upstream of the Lk6 gene. The Lk6 gene was originally identified biochemically as a microtubule-binding protein. LK6 localizes to centrosomes in the early blastoderm embryo and appears to be expressed ubiquitously throughout development. Loss-of-function mutations in Lk6 are viable and have no visible adult phenotype. Constitutive overexpression of Lk6 under the ubiquitin promoter causes microtubule defects in both eggs and embryos; these defects include fragmented mitotic spindles, abnormal asters, and abnormal metaphases, suggesting that LK6 may stabilize microtubules in vivo. Overexpression of phenotypes in the embryo suggests the same. Overexpression of Lk6 does not appear to affect imaginal tissues. The identification of a cytoskeleton-associated kinase that can genetically interact with the RAS pathway is consistent with studies in mammalian cells that indicate that the activation of the RAS/MAPK pathway can lead to spindle instability. Activated RAS induces chromosome aberrations such as dicentrics, acentrics, and double minutes. Genomic instability as measured by micronucleus formation increases tenfold in cells expressing activated RAS. Micronucleus formation induced by activated MAPK is due largely to disruption of the mitotic spindle rather than double-strand chromosome breaks (Huang, 2000 and references therein).
In the experiments described here, the overexpression of Lk6 in the developing eye has no visible phenotype. However, when overexpressed in the sev-RAS1V12 background, Lk6 is able to partially suppress the associated rough eye by reducing the number of supernumerary R7 cells. Since the supernumerary R7 cells in the sev-RASV12 background are generated postmitotically by cone cell to R7 transformations, in this instance LK6 may be playing a role in differentiation rather than proliferation. LK6 may be redundant with other kinases expressed in the eye, which normally act at other stages of development, or activate normally inactive signaling pathways when overexpressed. It is possible that expressing LK6 in the sev-RASV12 background could have a deleterious effect on cell differentiation and cause the improperly fated photoreceptors to die (Huang, 2000).
The MESR1 interactor identified in this screen is an insertion upstream of a novel gene that encodes a protein containing a rel domain. Sequence comparison of the rel domain with other known proteins of this class identifies it as a distant member of the NFAT family of transcription factors with mammalian NFAT5 its closest relative. NFAT1-4 are highly regulatable transcription factors that are sequestered in the cytoplasm of resting cells of the mammalian immune system. Upon activation of Calcineurin by intracellular calcium release, NFAT1-4 translocate to the nucleus where they bind cooperatively to DNA with the AP-1 heterodimers, FOS and JUN. The mammalian NFAT5 protein is distantly related to the others in sequence homology as well as function. NFAT5 appears to be constitutively nuclear, does not seem to interact with FOS and JUN, and does not transactivate well-characterized NFAT1-4 target promoters; its function and specificity remain unclear. Overexpression of this gene in the eye suppresses an activated RAS1 rough eye, implying that it acts to decrease RAS signaling efficiency. This protein may act to promote a nonneuronal cell fate or actively repress neuronal differentiation. There are four putative MAPK phosphorylation sites in the Drosophila NFAT5 homolog but it is unknown whether any of these sites are phosphorylated in vivo. No loss-of-function mutations have been identified to date. This protein may bind to RAS-dependent promoters -- identification of target genes will help elucidate the wild-type function of this gene in flies (Huang, 2000).
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date revised: 10 February 2010
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