The fission yeast homolog of the human transcription factor EAP30 blocks meiotic spindle pole body amplification.

Centrosome aberrations caused by misregulated centrosome maturation result in defective spindle and genomic instability. Here we report that the fission yeast homolog of the human transcription factor EAP30, Dot2, negatively regulates meiotic spindle pole body (SPB, the yeast equivalent of centrosome) maturation. dot2 mutants show excess electron-dense material accumulating near SPBs, which we refer to as aberrant microtubule organization centers (AMtOCs). These AMtOCs assemble multipolar spindles, leading to chromosome missegregation. SPB aberrations were associated with elevated levels of Pcp1, the fission yeast ortholog of pericentrin/kentrin, and reducing pcp1(+) expression significantly suppressed AMtOCs in dot2-439 cells. Our findings, therefore, uncover meiosis-specific regulation of SPB maturation and provide evidence that a member of the conserved EAP30 family is required for maintenance of genome stability through regulation of SPB maturation. EAP30 is part of a transcription factor complex associated with acute myeloid leukemia, so these results may have relevance to human cancer.

Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens.

BACKGROUND: Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. Spironucleus vortens is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of S. vortens is in progress, yet little information is available regarding molecular and cellular aspects of S. vortens biology beyond descriptive ultrastructural studies. To facilitate the development of S. vortens as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This construct also contains selectable antibiotic resistance markers for both S. vortens and E. coli. RESULTS: Stable transformants of S. vortens grew relatively rapidly (within 7 days) after electroporation and were maintained under puromycin selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under transcriptional control of the S. vortens histone H3 promoter, and visually confirmed diffuse GFP expression in over 50% of transformants. Next, we generated a histone H3::GFP fusion using the S. vortens conventional histone H3 gene and its native promoter. This construct was also highly expressed in the majority of S. vortens transformants, in which the H3::GFP fusion localized to the chromatin in both nuclei. Finally, we used fluorescence in situ hybridization (FISH) of the episomal plasmid to show that the transformed plasmid localized to only one nucleus/cell and was present at roughly 10-20 copies per nucleus. Because S. vortens grows to high densities in laboratory culture, it is a feasible diplomonad from which to purify native protein complexes. Thus, we also included a TAP tag in the plasmid constructs to permit future tagging and subsequent purification of protein complexes by affinity chromatography via a two-step purification procedure. CONCLUSION: Currently, progress in protistan functional and comparative genomics is hampered by the lack of free-living or commensal protists in axenic culture, as well as a lack of molecular genetic tools with which to study protein function in these organisms. This stable transformation protocol combined with the forthcoming genome sequence allows Spironucleus vortens to serve as a new experimental model for cell biological studies and for comparatively assessing protein functions in related diplomonads such as the human intestinal parasite, Giardia intestinalis.

A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish.

Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.