Ornithine decarboxylase antizyme
Northern analysis has revealed that the guf gene is expressed in all stages of embryonic and larval development as well as in pupae and adults. An abundant 2.1-kb transcript is detected in all developmental stages. An additional transcript (-1.6 kb) that labels faintly with guf probes may correspond to a different splicing variant of guf. However, this band could not be detected in other RNA samples prepared from similar developmental stages, suggesting that it may be a degradation product of the 2.1-kb transcript (Salzberg, 1996).
The expression pattern of the guf gene was determined with guf antisense RNA probes using in situ hybridization to whole mount embryos. Low levels of uniformly distributed guf RNA are detected in precellularized embryos, suggesting a maternal contribution of guf transcripts. During gastrulation and germband extension guf transcripts are mostly abundant in the mesoderm and the invaginating posterior midgut. At stage 11, patches of mesodermal cells that, based on their position, probably correspond to fat body precursors begin to express higher levels of guf RNA. A strong signal is also detected at this stage in the Malpighian tubule rudiments and the foregut. As germband retraction takes place, guf expression is maintained in the developing fat body, Malpighian tubules and the posterior and anterior midgut. In addition, at late stage 12 and stages 13-14 guf is expressed at low levels throughout most of the embryo including the subectodermal layer that contains most of the PNS cells. At stage 14, guf transcripts are first detected in the developing body wall muscles. Expression of guf in body wall muscles, fat body and the midgut is maintained throughout the rest of embryonic development (Salzberg, 1996).
The P-element enhancer detectors inserted in the guf gene confer ß-gal expression in a pattern similar to the distribution of guf mRNA. Very high levels of ß-gal activity were detected in the midgut (Kania, 1995). (Due to the strong expression in the gut and the defects in the sensory nervous system caused by these insertions the gene was named gutfeeling). Very weak lacZ expression was detected in the developing somatic muscles starting at stage 14 of embryonic development. Embryos heterozygous for these insertions display weak ß-gal expression in developing muscles, whereas expression in other tissues mentioned above is undetectable (Salzberg, 1996).
Since guf is expressed in Malpighian tubules and fat bodies, the Malpighian tubules and fat bodies of gut minus embryos were examined to determine whether they exhibit morphological defects. Malpighian tubules were visualized by anti-Cut staining and found to be morphologically normal in mutant embryos. Fat bodies were visualized with antibodies against Neurotactin and no obvious morphological defects were observed (Salzberg, 1996).
To assess the extent of maternal contribution of guf mRNA and to determine whether zygotic guf expression is eliminated in a deletion allele, mutant embryos were examined by in situ hybridization with guf antisense RNA probe. This probe did not contain sequences from the 5' terminus that were not removed by this deletion (the probe corresponds to nucleotides 1310-2164 of the cDNA). These experiments revealed that guf mRNA is present in guf homozygous mutant embryos until cellular blastoderm at levels similar to those found in wild-type embryos. The store of maternal guf mRNA is depleted rapidly during gastrulation, and by the end of stage 7 only low levels of residual guf transcripts are detected in the invaginating posterior midgut. No guf expression was detected in guf embryos at later stages of embryogenesis. These data suggest that zygotic guf expression is eliminated in guf mutant embryos (Salzberg, 1996).
If Sxl is a negative regulator of guf, it was reasoned that a correlation might be detectable between the expression of guf RNA and Sxl protein. RNA in situ hybridizations using guf sequences as a probe on wild-type ovaries has shown that the expression of guf is dynamic and changes considerably as the germ cells mature. Early germ cells have very low levels of guf RNA, but further down the germarium, the levels increase. guf RNA is also detected in the follicle cells of the germarium. In stage 2 egg chambers (the first chamber past the germarium), expression decreases to very low levels in both the follicle and germ cells. After stage 2, guf RNA is expressed in the germ cells but is essentially undetectable in the follicle cells. As vitellogenesis begins, the levels in germ cells drop again and guf RNA is detected primarily in the oocyte. After this stage, high levels are seen in the nurse cells and oocyte, while follicle cell expression remains very low (Vied, 2003).
The cells in the germarium that show very low levels of guf RNA are the early germ cells, which also express high levels of cytoplasmic Sxl. guf RNA levels begin to increase in the region of the germarium where Sxl is downregulated, suggesting that Sxl might act negatively on guf. By contrast, in Sxlf4 ovaries, guf RNA levels are found to be high throughout, including the germ cells immediately below the terminal filament. An increase in guf RNA is also seen in Sxlf5 ovaries. These observations support the hypothesis that, normally, Sxl downregulates guf expression and suggest that the mutant Sxlfs proteins are inefficient at this function. Consistent with the observation that decreasing the dose of guf partially rescues the Sxlfs female sterile phenotype, wild-type levels of guf RNA are seen in the germ cells of Sxlf5/Sxlf1;guf118-3/+ ovaries (Vied, 2003).
The gutfeeling (guf) gene was uncovered in a genetic screen for genes that are required for proper development of the embryonic peripheral nervous system. Mutations in guf cause defects in growth cone guidance and fasciculation and loss of expression of several neuronal markers in the embryonic peripheral and central nervous systems. guf is required for terminal differentiation of neuronal cells. Mutations in guf also affect the development of muscles in the embryo. In the absence of guf activity, myoblasts are formed properly, but myoblast fusion and further differentiation of muscle fibers is severely impaired. The guf gene was cloned and found to encode a 21-kD protein with a significant sequence similarity to the mammalian ornithine decarboxylase antizyme (OAZ). In mammals, OAZ plays a key regulatory role in the polyamine biosynthetic pathway through its binding to, and inhibition of, ornithine decarboxylase (ODC), the first enzyme in the pathway. The elaborate regulation of ODC activity in mammals still lacks a defined developmental role and little is known about the involvement of polyamines in cellular differentiation. Guf is the first antizyme-like protein identified in invertebrates (Salzberg, 1996).
Mutations in the gutfeeling, gene were uncovered in a genetic screen designed to identify genes that play a role in the development of the embryonic PNS (Kania, 1995). Embryos from 2000 strains carrying homozygous lethal P-element insertions on the second chromosome were stained with monoclonal antibody (MAb) 22C10 and examined for defects in the PNS. The guf gene was identified by three insertional mutations that cause similar phenotypes and were mapped to cytological position 48E5-12. Precise or near precise excision of a P-element insertion in guf reverts both the lethality and the PNS phenotype, demonstrating that the insertion causes the phenotype. Additional guf alleles were generated by imprecise excision of a P-element insertion. One allele corresponds to a small deficiency that removes most of the guf transcription unit and the entire open reading frame (Salzberg, 1996).
This deletion was found to abolish zygotic expression of guf and hereafter is referred to as a null allele. The insertional alleles cause phenotypes that are very similar to those observed in guf null embryos, suggesting that they are severe loss of function or null alleles (Salzberg, 1996).
Mutations in guf are embryonic lethal and cause multiple defects in the embryonic PNS. A smaller number of PNS neurons express 22C10 in homozygous guf embryos than in wild-type embryos and the overall intensity of 22C10 immunoreactivity is much decreased when compared to wild-type embryos (Kania, 1995). The lateral and ventral PNS clusters are more severely affected than the dorsal and ventral clusters. On average, six to seven neurons are labeled with MAb 22C10 in lateral clusters of guf embryos as compared to 10-11 in wild-type, and only three to four neurons label in the ventral cluster of mutant embryos as compared to seven in wild-type. The lateral chordotonal neurons seem to be most severely affected by mutations in guf and only one to three chordotonal neurons are evident in each lateral cluster as compared to five in wild-type embryos. However, the loss of 22C10 immunoreactivity is not restricted to chordotonal neurons and is evident in all types of sensory neurons (Salzberg, 1996).
In addition to the lack of 22C10 expression, PNS axons of guf embryos display severe defects in growth cone guidance and fasciculation. One of the most noticeable defects that is observed in all guf embryos is splitting of the intersegmental nerve (ISN) into two fascicles. The ISN 'loop' phenotype was observed in 57% of abdominal segments in guf homozygous mutant embryos. Crossing of the ISN into neighboring segments is much less frequent and was observed in single segments of -40% of the examined guf null embryos. Defects in growth cone guidance and fasciculation are not restricted to ISNs but are also evident in segmental nerves (Salzberg, 1996).
MAb 22C10 is a relatively late marker for PNS development, since neuronal cells express the 22C10 antigen only after they exit the cell cycle and start differentiating as neurons. Thus, lack of 22C10 expression may indicate a true loss of neurons stemming from a failure of precursor formation, failure of precursor division, transformation of neurons into lineage-related support cells, or cell death. Alternatively, this phenotype may reflect a failure of neuronal cells to fully differentiate and express late neuronal markers. To distinguish among these possibilities, a variety of cell-type-specific markers were used to assess number and identity of PNS cells in guf embryos of various developmental stages (Salzberg, 1996).
Since chordotonal neurons are among the most severely affected neurons the analysis focused on the formation of the conspicuous cluster of lateral chordotonal organs. To determine whether the precursors of chordotonal organs were affected in guf embryos, they were immunocytochemically stained with antibodies against the Atonal protein. The pattern of chordotonal SOPs in the mutant was found to be indistinguishable from that of wild-type embryos. To determine whether SOP division is impaired, the PNS of mutant embryos was examined with anti-Couch potato (Cpo) antibodies. The Cpo protein is present in nuclei of all the cells that constitute each sensory organ. Thus, the number of CPO positive cells reflects the number of cell divisions each precursor went through. The level of CPO protein is reduced in guf mutant embryos when compared to wild-type, but a wild-type number of CPO positive nuclei is evident in each chordotonal organ (Salzberg, 1996).
These observations indicate that zygotic guf is not required for the determination of SOPs or their division but is required during later stages of PNS development. Loss of 22C10 immunoreactivity may reflect a transformation of chordotonal neurons into another type of neurons that normally express lower levels of this antigen. Hence, embryos were stained with anti-Cut antibody that labels only external sensory organs and a subset of multiple dendritic neurons appraisal of whether the identity of chordotonal neurons is altered in guf mutants. The pattern of Cut expression appears normal in guf embryos, indicating no change in the identity of sensory organs. To determine whether the identity of cells within each chordotonal organ is altered, embryos were stained with anti-Prospero to reveal sheath cells, and the anti-Repo/RK2 antibody to reveal ligament cells. Normal numbers of sheath and ligament cells are evident in most lateral chordotonal organs of guf embryos. The results of these experiments suggest that loss of guf activity does not alter the identity of sensory organs, nor does it alter the identity of cells within each organ. It is concluded that guf is not required for cell fate determination but rather for terminal differentiation of the developing PNS cells (Salzberg, 1996).
Whereas cell type-specific antigens are expressed in a normal spatial pattern in the PNS of guf mutants, the level of many markers is greatly reduced. Reduced expression levels were observed for the 22C10 antigen and CPO protein in mature embryos. In addition, the level of the glial homeo protein Repo in ligament cells of the lateral chordotonal organs, but not in exit and peripheral glia, is severely decreased when compared to wild-type embryos. However, the levels of Prospero and Cut proteins in the PNS seem to remain unaffected by mutations in guf. These observations indicate that mutations in guf may exert their effect on late PNS differentiation by altering protein levels (Salzberg, 1996).
Since mutations in guf seem to affect most or all types of sensory neurons, the CNS of mutant embryos was examined to determine whether CNS neurons are affected in a similar fashion. guf embryos exhibited a severe reduction in the number of 22C10-expressing cells in the ventral nerve cord when compared to wild- type embryos. However, the number of neuroblasts in the CNS of stage 10-11 guf embryos appeared normal when visualized with antibodies against Prospero. The overall width of the ventral nerve cord appeared normal when examined with Nomarski optics. This suggests that as in the PNS, the lack of 22C10 expression in the CNS does not reflect a true loss of neuronal cells but a failure of these cells to fully differentiate as neurons (Salzberg, 1996).
To further assess late neuronal differentiation in the CNS, embryos were stained with antibodies against the synaptic vesicle protein Synaptotagmin. This protein serves as a terminal differentiation marker, as expression and synaptic localization of Synaptotagmin just precedes synaptic activity. Synaptotagmin is barely detectable in guf embryos when compared to wild type. This observation again supports the view that mutations in guf impair late neuronal differentiation by altering protein levels (Salzberg, 1996).
To examine the axonal pathways in the CNS of guf mutants, embryos were immunohistochemically stained with MAb 1D4 that recognizes the Fasciclin-II (FasII) protein. In wild-type embryos, anti-FASII staining reveals three distinct longitudinal tracts on either side of the midline. The organization of these tracts in guf embryos is aberrant. Although three distinct fascicles can be identified in some segments, most often only two fascicles are evident and these fascicles appear less tight than normal. The most medial fascicle is the least severely affected, whereas the most lateral fascicle is the most severely affected. In addition, the posterior commissure appears thicker than normal (Salzberg, 1996).
In summary, the defects observed in the CNS of guf embryos are very similar to those observed in the PNS and consist of alterations in the protein levels of neuronal-specific markers coupled to misrouting of axons and defects in fasciculation. These data suggest that guf plays a similar role in PNS and CNS development. However, further analysis of cell identities in the CNS of guf mutants is required to exclude the possible involvement of guf in cell fate determination in the CNS (Salzberg, 1996).
Examination of guf embryos with Nomarski optics indicates that mutant embryos also display defects in muscle development. To study the fully developed muscle pattern, embryos were generated with antibody against Drosophila muscle Myosin. This antibody labels all somatic muscle fibers, the cardial cells and the visceral muscles. Somatic muscles of the body wall are organized in a highly stereotyped pattern in wild-type embryos. This pattern is considerably disrupted in guf homozygous mutant embryos where numerous muscles are missing, displaced or exhibit aberrant shapes (Salzberg, 1996).
In addition, these embryos contain many mononucleate myoblasts that failed to fuse into muscle syncytia. Similar defects were observed in guf mutant embryos stained with antibodies against the spectrin-like MSP-300 protein that is expressed in muscles and chordotonal neurons. The level of MSP-300 protein is greatly reduced in muscle fibers and chordotonal neurons of mutant embryos when compared to wild type (Salzberg, 1996).
Anti-Myosin staining also revealed defects in the visceral and cardiac musculature. In wild-type embryos, a thin layer of evenly spread visceral muscles is detected around the gut. In guf embryos the visceral muscles seem to spread properly around the developing gut, however the thickness of this muscle layer appears less uniform than that of wild-type embryos leading to its rippled appearance. The constrictions of the gut form properly in guf mutants. The contractile part of the heart normally consists of a double row of Myosin expressing cardial cells that form a linear tube underneath the dorsal midline. Although the cardial cells form properly and the assembly of the heart into a linear tube appears relatively normal in guf mutants, Myosin expression in the cardial cells is essentially absent (Salzberg, 1996).
To determine whether the defects observed in the somatic musculature of guf embryos stem from aberrant specification of these muscles or from later differentiation defects, guf embryos were stained for nautilus RNA and for the Evenskipped (Eve) protein. nautilus is normally expressed in a subset of developing muscles in stage 12/13 embryos. This expression pattern does not appear to be altered in guf embryos. The Eve protein is also present in the nuclei of the dorsal-most muscle and pairs of pericardial cells in each abdominal segment. In guf mutants, the number of Eve positive cells appears similar to wild type, but the distribution of nuclei in the dorsal somatic muscle is abnormal. Instead of segregating into two crescent rows of nuclei on either side of the segment, these nuclei remain clumped together. Similarly, these dorsal muscles display abnormal morphology when visualized with anti-Myosin antibody and do not appear as broad and spread out as in wild type. The aberrant distribution of nuclei within muscle fibers is not restricted to the dorsal-most muscles and is evident in all muscle fibers when stained with antibodies against Mef2. These results indicate that the initial specification of individual muscles occurs properly in the absence of guf activity, but myoblast fusion and distribution of the syncytial nuclei within the developing muscle fibers is aberrant. Hence, it has been concluded that in the myogenic lineages, as in the PNS, guf is not required for cell type specification but for terminal differentiation of muscle cells. It is proposed that mutations in guf affect muscle development by reducing the level of various muscle-specific proteins (Salzberg, 1996).
These data suggest that guf plays a role in terminal differentiation of PNS cells following their specification as neurons or support cells but is not required for the specification of cell identities. Based on the observation that the levels of several neuronal-specific proteins are abnormally low in mutant embryos, it is proposed that guf is required for the regulation or maintenance of gene expression in the differentiating PNS. The failure of PNS cells to express late neuronal markers may explain the abnormal morphology of neuronal cells and the defects in growth cone guidance and fasciculation (Salzberg, 1996).
The pattern of body wall muscles is also disrupted in guf mutant embryos since many muscle fibers are missing or misplaced. In addition, the layer of visceral muscles surrounding the gut appears rippled and less regular than normal. Early subdivision of the mesoderm to somatic, visceral and cardiac mesoderm was found to occur normally in the absence of guf, and the initial specification of individual muscles does not seem to be affected. However, myoblast fusion and segregation of the syncytial nuclei within the developing muscles is aberrant leading to abnormal morphology or complete loss of specific muscle fibers. Based on these data it has been proposed that guf is required for differentiation but not cell fate determination of all types of embryonic muscles (Salzberg, 1996).
To determine whether guf has a function in female germ cell development, germ cell clones were generated that were homozygous mutant for guf. Clones were induced by using the FLP/FRT mitotic recombination system in a background that had the SmD3+ genomic transgene, to preclude effects of this gene. When induced during the larval stage, clones of germ cells mutant for guf were not recovered in adult ovaries, suggesting a requirement for guf in the early stages of germ cell development. When induced in adults, germ cell clones did not develop beyond stage 2 egg chambers. Within 7 days of induction, many guf mutant cysts were observed in the germarium, and these showed no obvious defects, including their expression of Sxl. However, 14 days after induction, no guf mutant cysts were detected in germaria, but multiple degenerating egg chambers that had been pinched off were observed. These data suggest that, in the adult ovary, guf is not required in the early stages of germ cell development but in the cysts past the germarium, and that guf is needed either for the survival or the prevention of differentiation of germline stem cells. Since guf mutant germ cells show near normal Sxl expression, the data indicate that loss of guf does not have a major effect on Sxl expression in the early dividing germ cells. The proposal that Sxl maintains guf at low levels in region 1 of the germarium is consistent with this view (Vied, 2003).
Germ cells mutant for guf appear to develop through the germarium normally. Therefore whether ectopic expression of guf would affect early germ cell development was examined. Overexpression of Guf causes a decrease in the levels of cytoplasmic Sxl, resulting in fewer germ cells with high levels of the protein under the terminal filament. This effect requires efficient expression of Guf since it is only observed when the frameshift-independent variant of guf, is used. This latter observation also confirms that Guf translation requires frameshifting in vivo (Vied, 2003).
As a different means of increasing Guf levels, wild-type ovaries were treated with polyamines (spermidine). Polyamines increase the translation of endogenous guf RNA by increasing the rate of translational frameshifting. Spermidine causes a decrease in the number of early germ cells with high levels of cytoplasmic Sxl. This effect was observed within 3 h, implying that the change in Sxl is not caused by differentiation of the germ cells. The possibility that spermidine affects Sxl by a Guf-independent mechanism cannot be excluded. However, it is likely that an increase in Guf is involved given the overexpression data above (Vied, 2003).
The effect of Guf overexpression on Sxl in early germ cells strongly resembles the effect of overexpressing the patterning gene, hh (Vied, 2001). In the ovary, Hh is expressed in the terminal filament and cap cells, the somatic cells at the tip of the germarium. Hh regulates the proliferation of the follicle cells of the ovary and has minor effects on germ cell development (Vied, 2003).
To test for an epistatic interaction between hh and guf, hs-hh; guf118-3/+ flies were generated. Removing one copy of guf counteracts the overexpression of Hh, and there is an increase in the number of early germ cells with high levels of cytoplasmic Sxl. Previous data (Vied, 2001) suggested that Hh regulates not only the levels but also the nuclear localization of Sxl in early germ cells. The coding sequence of Sxl suggests a leucine-rich Nuclear Export Signal (NES). Whether the Sxl NES is functional was tested using leptomycin B (LMB). LMB prevents the nuclear export of proteins by binding to exportin 1/CRM1, which binds directly to leucine-rich NES sequences. Wild-type ovaries treated with LMB show an increase in detectable nuclear Sxl. By contrast, early germ cells that have not been treated with LMB show no distinct nuclear foci of Sxl. Consistent with Hh affecting Sxl nuclear entry, overexpression of Hh in the presence of LMB significantly increases the amount of nuclear Sxl. Overexpression of Guf in the presence of LMB has a similar effect, and the effect of Guf is usually stronger than that of Hh (Vied, 2003).
To demonstrate that guf acts downstream of Hh in Sxl nuclear entry, Hh was overexpressed in a background with a reduced dose of guf (hs-hh; guf118-3/+) and the ovaries were treated with LMB. As was seen for the degradation of Sxl, removal of one copy of guf counteracts Hh-regulated nuclear entry of Sxl, and Sxl remained predominantly in the cytoplasm. This observation suggests that Guf functions downstream of Hh in both nuclear entry and degradation, promoting the release of cytoplasmic Sxl in early germ cells (Vied, 2003).
Besides Sxl, Hh affects other germ cell-specific processes. Hh overexpression decreases the ß-Spectrin staining of the fusome. ß-Spectrin is one of the proteins associated with the fusome, a germline specific organelle that plays a role in establishing the interconnected 16-cell cyst. Overexpression of Guf does not appear to affect the fusome. Additionally, reducing the dose of guf does not reverse the effect of Hh overexpression on the fusome. These data implicate guf as a downstream effector of Hh, but only in regulating Sxl (Vied, 2003).
In Drosophila, Cyclin B is not essential for viability but is necessary for female fertility. Since Sxl has been shown to be important for the mitosis of early germ cells, a correlation between Sxl and Cyclin B was sought. In early germ cells, Cyclin B colocalizes with Sxl and is downregulated concomitantly with Sxl. Since Sxl and Cyclin B colocalize in early germ cells, the effect of Hh on Cyclin B was examined. Overexpression of Hh results in fewer Cyclin B-expressing early germ cells, as seen for Sxl. Furthermore, the germ cells that express Cyclin B show reduced levels of the protein. This similarity in response prompted a test to see whether Hh also regulates the nuclear entry of Cyclin B. Overexpression of Hh and treatment with LMB results in higher levels of nuclear Cyclin B than in wild-type early germ cells treated with LMB only. This observation suggests that Hh promotes the nuclear entry of Cyclin B (Vied, 2003).
Since Guf appears to act downstream of Hh to regulate Sxl, whether Cyclin B would also respond to Guf was also examined. Ectopic expression of Guf has the same effect on Cyclin B as on Sxl. Fewer early germ cells with cytoplasmic Cyclin B were observed, and Sxl and Cyclin B continued to colocalize. LMB treatment with Guf overexpression increases the nuclear levels of Cyclin B. As for Sxl, Cyclin B responds more strongly to Guf than Hh (Vied, 2003).
To determine whether Guf also acts downstream of Hh in affecting Cyclin B nuclear entry, hs-hh; guf118-3/+ ovaries treated with LMB were examined. Under these conditions, Cyclin B was predominantly cytoplasmic, suggesting that Guf also functions downstream of Hh in the regulation of Cyclin B (Vied, 2003).
In vertebrates, the intracellular localization of Cyclin B is critical to its function. An NES within the cytoplasmic retention signal (CRS) allows Cyclin B to rapidly shuttle out of the nucleus. Phosphorylation of the CRS results in nuclear accumulation of Cyclin B and its associated Cyclin-dependent kinase with which Cyclin B initiates mitosis. Since Sxl and Cyclin B appear to undergo similar changes in response to Hh and Guf, tests were made to determine whether Cyclin B is affected by changes in Sxl.
Cyclin B was examined in ovaries with mutant Sxl protein. Like Sxl, Cyclin B is localized to the cytoplasm of Sxlf4 germ cells. Treatment of Sxlf4 ovaries with LMB caused the Sxl, but little Cyclin B, protein to accumulate in the germ cell nuclei. As overexpression of Hh with LMB treatment increases the nuclear levels of both Sxl and Cyclin B in wild-type ovaries, the intracellular localization of both proteins was examined in Sxlf4 ovaries after similar treatment. The mutant Sxl protein accumulates in the nuclei, but surprisingly, Cyclin B remains in the cytoplasm. Consistent with this observation, ovaries doubly homozygous for Sxlf4 and Su(fu) treated with LMB show nuclear accumulation of Sxlf4 protein, but not Cyclin B. These results suggest that, normally, Sxl affects the rate of nuclear entry of Cyclin B. Interestingly, the mutations in the Sxlfs alleles alter a region of the protein that is proline-rich. Proline-rich domains have been described as being involved in protein-protein interactions (Vied, 2003).
Sxl can enter the nucleus without the concomitant entry of Cyclin B. Cyclin B mutant ovaries were examined to test the assumption that Sxl does not require Cyclin B for nuclear exiting. A null mutation for cyclin B derived from the imprecise excision of a P-element in the 5' UTR (cycB3) is not lethal except when combined with other cell cycle regulators. The cycB3 mutation results in female sterility with many agametic ovarioles. When germ cells were present, the localization of Sxl was primarily cytoplasmic. Treatment with LMB shows that Sxl is able to enter the nucleus at levels comparable to those observed in wild-type ovaries\. These results suggest that Sxl does not require Cyclin B for nuclear exiting. Otherwise, Sxl would be primarily nuclear in the absence of Cyclin B (Vied, 2003).
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date revised: 12 May 2003
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