To identify proteins that may form complexes with Cyclin J, the yeast two-hybrid system was used to screen two Drosophila cDNA libraries, one from ovaries and one from embryos. In addition to Cdk2, three other Cyclin J-interacting proteins were isolated from the libraries. Full-length versions of the DNA supercoiling factor Scf (Kobayashi, 1998) and of a putative GTPase, Arl2 (Clark, 1993), and the carboxy-terminal 371 amino acids of S5a, the non-ATPase subunit of the 26S proteasome (Haracska and Udvardy, 1995), were isolated. Each of these proteins, as well as the Cdk subunit Cks1 (FBgn0010314, which previously had been shown to bind to Cyclin J (Finley, 1994), interacts specifically with Cyclin J but not with a panel of unrelated proteins in the two-hybrid assay. No full-length Cdks other than Cdk2 were isolated. Moreover, in direct tests, Cyclin J failed to interact with other Cdks, including Cdk1, Cdk4, and Cdk5. These results suggested that Cdk2 may be the kinase partner for Cyclin J (Kolonin, 2000).
To determine whether Cdk2 interacts with Cyclin J in Drosophila, affinity-purified Cyclin J antibodies were used to precipitate Cyclin J complexes from embryo extracts. The precipitates were subjected to immunoblotting with Cdk1 or Cdk2 antibodies. Consistent with the two-hybrid interactions, Cdk2 and Cyclin J coimmunoprecipitate from unfertilized eggs and syncytial embryos. The Cyclin J complexes contain both of the previously described phosphoisoforms (Sauer, 1995) of Cdk2. No coimmunoprecipitation of Cyclin J and Cdk2 has been detected from older embryos, except from embryos ectopically expressing Cyclin J from the hs>CycJ transgene. Cdk1 is not detected in complexes with Cyclin J in early embryos. However, Cdk1 was detected in Cyclin J immunoprecipitates from unfertilized eggs and from older hs>CycJ embryos. Combined, these results suggest that Cyclin J can bind both Cdk1 and Cdk2 in vivo and that Cdk2 is the principal kinase subunit for Cyclin J in the syncytial embryo (Kolonin, 2000).
To determine whether Cyclin J is associated with an active kinase in embryos a test was performed to see whether Cyclin J immunoprecipitates can phosphorylate histone H1. Cyclin J-associated H1 kinase activity can be detected in newly laid unfertilized eggs and embryos 0-4 h AED. The activity diminishes between 3 and 6 h and after 6 h reaches the background level that can be immunoprecipitated with preimmune serum. Precipitation of the Cyclin J-associated kinase activity from both syncytial embryos and later embryos that ectopically express Cyclin J can be blocked by CycJc peptide, but not by a control peptide, PepC2. In contrast, CycJc does not block immunoprecipitation of cyclin B-associated H1 kinase activity or the background activity. These data, combined with the results from coimmunoprecipitations and two-hybrid interaction assays, suggest that early embryos contain active Cdk2/Cyclin J complexes that are inactivated around the time of cellularization by degradation of Cyclin J (Kolonin, 2000).
To determine whether Cyclin J protein is present during early embryogenesis, rabbit antibodies raised against the unique C-terminal 100 amino acids of Cyclin J (CycJc) were used. Affinity-purified Cyclin J antibodies recognize a single protein of the appropriate molecular weight (~50 kDa) in immunoblots from early embryos. The abundance of this protein diminishes during embryogenesis, yet can be dramatically induced in older embryos that carry a heat-shock-inducible Cyclin J transgene (hs>CycJ). The Cyclin J protein was abundant in unfertilized eggs and in embryos 0-1.5 h after egg deposition (AED), but diminishes to a low level by 3 h AED. A similar pattern is observed by immunoprecipitating proteins from embryo extracts using the affinity-purified rabbit Cyclin J antibodies and probing immunoblots using a mouse antisera raised against CycJc; Cyclin J protein is detected in unfertilized and 0- to 3-h-old embryos, but cannot be detected in older embryos. Under the conditions used to stage these embryos, cellularization is completed between 2 and 3 h AED. Thus, Cyclin J is most abundant in the syncytial embryo. This pattern is in contrast to the expression profile of cyclin B, which is abundant throughout embryogenesis, and of cyclins A, B3, and E, which are expressed in a temporal pattern similar to that of cyclin B. Consistent with the immunoblots, the Cyclin J antibodies strongly stain wild-type embryos undergoing the first 10 divisions. Starting at late syncytial stages, embryo staining decreases to a background level. Cyclin J staining can be induced in later embryos that contain the hs>CycJ transgene or in a striped pattern in embryos expressing CycJ under control of the prd promoter. No obvious subcellular localization is observed in the early embryos or hs hs>CycJ embryos. In adult Drosophila Cyclin J staining is strong in the cytoplasm of the ovarian nurse cells, but is not detected in testes in males. Combined, these data suggest that Cyclin J protein is deposited into eggs maternally and is degraded during early embryogenesis (Kolonin, 2000).
Northern analysis shows that Cyclin J expression is strictly maternal; its mRNA is present in the newly laid egg and in adult females but is undetectable in the embryo after zygotic transcription begins. This expression pattern suggests that cyclin J may be involved in the early nuclear division cycles that lack G1 and G2 phases. Alternatively, cyclin J may function in the ovary during oogenesis (Findley, 1996),
Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. This study assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis.These results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised (Atikukke, 2014).
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date revised: 28 January 2015
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