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Rosiglitazone maleate mg Our outcrossing data confirmed that
Our outcrossing data confirmed that ACL1 and ACL2 have important temporal and spatial functions in ascospore delimitation in G. zeae. When each ACL deletion mutant was used as male for outcrossing, nuclear division and spore delimitation were not properly progressed in most asci. Ascospores that contained multiple nuclei were frequently observed and were much larger in size than ascospores with only one nucleus, indicating that the size variation in ascospores produced by these crosses was a result of the increased number of nuclei. In the pseudohomothallic fungus Neurospora tetrasperma, four binucleate spores usually formed after two meiotic divisions and a single mitosis; in some cases, however, ascospores were enclosed with a single nucleus. Some ascospores were delimitated with multiple nuclei in the Neurospora crassa Fsp-1 mutant (Raju, 1992). Consistent with our observations, the ascospores with multiple nuclei were bigger than the ascospores with a single nucleus in both cases. In mammals, a single ACL gene exists, but plants, fungi, and some prokaryotes have two genes coding for two ACL sub-units, both of which are required for ACL activity (Fatland et al., 2002, Kanao et al., 2001, Nowrousian et al., 1999). In this study, single and double deletion mutants had similar phenotypes. However, ACL1 deletion caused more severe defect in ascospore formation than ACL2 deletion, when the deletion mutants were used as a male (Fig. 4), suggesting that the two genes perform distinct and non-redundant roles in these processes. This difference also may arise from haploinsufficiency in young asci. G. zeae has a short diploid phase in young asci during sexual reproduction. The diploid cells may require certain levels of ACL1 and ACL2 for proper sexual reproduction, and the presence of a single functional ACL1 in the cells may cause more severe haploinsufficiency than that of ACL2. The low level of histone acetylation in ACL deletion mutants may be responsible for the defects in sexual reproduction. For orderly nucleosome assembly to occur, newly synthesized histones have to be rapidly acetylated by histone acetyltransferases that catalyze the transfer of an acetyl moiety from acetyl-CoA to lysine residues. This process occurs when DNA is replicated during the synthesis (S) phase of the Rosiglitazone maleate mg (Lucchini and Sogo, 1995, Ruiz-Carrillo et al., 1975, Shahbazian and Grunstein, 2007, Verreault, 2000). Since the regulation of histone proteins and DNA synthesis is delicately linked, a slight error in these processes could result in DNA damage or even cell death (Bird et al., 2002, Gunjan et al., 2005). Histone acetylation is also important for a broad range of transcriptional regulation through the folding of chromatin. After positively charged histones are acetylated, the charge is neutralized, which results in a decreased interaction between DNA and histones and provides binding sites for proteins involved in transcription. Inappropriate acetylation of histones leads to defects in global gene regulation (Wellen et al., 2009). Also in the filamentous fungus A. nidulans, removal of heterochromatin marks through histone acetylation or methylation by velvet complex was thought to activate developmental related genes (Reyes-Dominguez et al., 2010, Shwab et al., 2007). Sexual reproduction in fungi needs a delicate regulation of hundreds of genes that are responsible for fruiting body formation, ascospore maturation, and the discharge of ascospores. Therefore, inaccurate transcriptional regulation of such genes due to a low level of histone acetylation during sexual reproduction may result in a complete loss of fertility. ACL mutants also had defects in asexual development. In mammalian cells, ACLY is crucial for the transcription of glycolytic genes through histone acetylation (Wellen et al., 2009). Histone acetylation is known to have a global role in mitotic gene expression in yeast (Krebs et al., 2000). In this study, the histone acetylation levels in both the wild-type strain and ACL mutants during vegetative growth were too low to make a meaningful comparison. Rapid histone acetylation and deacetylation as well as the overall level of histone acetylation are important regulatory process in the regulation of cellular functions (Waterborg, 2002). The defects in asexual development of ACL deletion mutants may be caused by improper histone acetylation and deacetylation, even during times when the histone acetylation levels are too low to measure. Another possibility is that the defects may be directly related to the reduction of cytosolic acetyl-CoA in the mutants. Exogenous treatment of acetate completely restored defects in asexual development but not sexual development (Table 2). In addition, the transcription profiles of ACL genes in G. zeae showed that the amount of transcripts were higher in asexual stage than in sexual stage when histone acetylation was high (Fig. 8). These results suggest that G. zeae needs more cytosolic acetyl-CoA during the asexual stage than sexual stage and that the defects are caused by a deficiency of cytosolic acetyl-CoA triggered by deletion of ACL genes.