The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex.

The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that coordinates progression through the cell cycle by temporally and spatially promoting the degradation of key proteins. Many of these targeted proteins have been shown to play important roles in regulating orderly progression through the cell cycle. Using a previously described Drosophila in vitro expression cloning approach, we screened for new substrates of the APC in Xenopus egg extract and identified Drosophila MCPH1 (dMCPH1), a protein encoded by the homolog of a causative gene for autosomal recessive primary microcephaly in humans. The dMCPH1-B splice form, but not the dMCPH1-C splice form, undergoes robust degradation in Xenopus interphase egg extract in a Cdh1-dependent manner. Degradation of dMCPH1-B is controlled by an N-terminal destruction box (D-box) motif as its deletion or mutation blocks dMCPH1-B degradation. dMCPH1 levels are increased in Drosophila morula (APC2) mutant embryos, consistent with dMCPH1 being an APC substrate in vivo. Using a purified, reconstituted system, we show that dMCPH1-B is ubiquitinated by APC(Cdh1), indicating that the effect of APC on dMCPH1-B ubiquitination and degradation is direct. Full-length human MCPH1 (hMCPH1) has been predicted to be an APC substrate based on its interaction with the APC subunit Cdc27. We were not able to detect changes in hMCPH1 levels during the cell cycle in cultured human cells. Overexpression of hMCPH1 (or dMCPH1-B) in developing Xenopus embryos, however, disrupts cell division, suggesting that proper regulation of hMCPH1 and dMCPH1-B activity plays a critical role in proper cell-cycle progression.

no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila.

In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ;no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.

The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo.

Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. We identified mcph1, the Drosophila homolog of MCPH1, in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. We propose a model in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development.

QIAamp MinElute virus kit effectively extracts viral nucleic acids from cerebrospinal fluids and nasopharyngeal swabs.

BACKGROUND: Nucleic acid preparation from a variety of clinical specimens requires efficient target recovery and amplification inhibitor removal and is critical for successful molecular diagnosis. The QIAamp MinElute Virus kit (Qiagen Inc., Valencia, CA) was compared to the two existing methods currently used in our laboratory, IsoQuick (Orca Research Inc., Bothell, WA) for DNA extraction and RNAzol B (Leedo Laboratories Inc., Houston, TX) for RNA extraction, of viral nucleic acids. STUDY DESIGN: A total of 150 clinical specimens, including cerebrospinal fluid (CSF) and nasopharyngeal swabs (NPS), were used to determine the extraction efficiency of the MinElute compared to the other two methods. Nucleic acid recovery, hands-on time, turn-around-time and cost were compared across all kits. RESULTS: There was complete concordance between the MinElute and IsoQuick/RNAzol kits when herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), influenza A virus or enteroviruses were detected using a colorimetric microtiter plate PCR system. The kits were equivalent in their ability to detect either DNA or RNA with superior ability to recover a high quality and quantity of RNA. With the potential to process larger specimen volumes, the MinElute kit can significantly shorten processing time from 2h to 50-55min. CONCLUSIONS: Although relatively high test kit costs were noted, the MinElute kit provides another rapid and user-friendly specimen processing tool in the diagnostic molecular microbiology laboratory.