Repeat-induced G-C to A-T Mutations in
            <i>Neurospora</i>

Cambareri, Edward B.; Jensen, Bryan C.; Schabtach, Eric; Selker, Eric U.

doi:10.1126/science.2544994

Cited by 354 publications

(272 citation statements)

References 11 publications

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“…Because the catalytic subunits of PP1 and PP2A (encoded for by the ppp-1 and pph-1 genes, respectively) are essential for cell survival in Neurospora and other eukaryotes (Yatzkan and Yarden 1995;Zeke et al 2003), we used repeat-induced point mutation (RIP; Cambareri et al 1989) to obtain partially functional To examine whether these mutations led to the decrease of the phosphatase activity of PP1, phosphatase assay (see supplemental materials) was performed using extracts prepared from cultures grown in LL, a condition that the clock is not running. The 32 P-labeled phosphorylase a is used as the substrate of the assay in the presence or absence of inhibitor-2, a specific protein inhibitor for PP1 (Krebs and Fischer 1962;Yatzkan et al 1998;Cohen 2002).…”

Section: Resultsmentioning

confidence: 99%

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Yang¹,

He²,

Cheng³

et al. 2004

Genes Dev.

111

117

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Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in a rgb-1 (a regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and longperiod oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, suggesting that these two phosphatases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.Supplemental material is available at http://www.genesdev.org.Received September 15, 2003; revised version accepted December 15, 2003. Reversible phosphorylation is an important regulatory mechanism for many biological processes in eukaryotic organisms. The phosphorylation state of a protein is controlled dynamically by protein kinases and phosphatases. Previous studies have demonstrated that phosphorylation of circadian clock proteins is an essential posttranscriptional mechanism in the regulation of circadian clocks (Dunlap 1999;Young and Kay 2001). Several kinases, including casein kinase I (CKI) and CKII, have been shown to phosphorylate and regulate the key clock components in eukaryotic systems Sugano et al. 1999;Lowrey et al. 2000;Gorl et al. 2001;Yang et al. 2002).FREQUENCY (FRQ), WHITE COLLAR-1 (WC-1), and WC-2 proteins are three key components in the Neurospora frq-wc-based circadian oscillator . In addition to being the circadian blue light photoreceptor (Froehlich et al. 2002;He et al. 2002), in the dark, WC-1 forms a heterodimeric complex with WC-2 through their PAS domains and activate frq transcription by binding its promoter (Crosthwaite et al. 1997;Talora et al. 1999;Cheng et al. 2001bCheng et al. , 2002Cheng et al. , 2003Froehlich et al. 2003). FRQ, the negative element of the feedback loop, inhibits its own transcription through interactions with the WC complex (Aronson et al. 1994;Cheng et al. 2001a;Denault et al. 2001;Froehlich et al. 2003). FRQ protein is immediately phosphorylated after its synthesis and becomes extensively phosphorylated prior to its degradation by the ubiquitin/proteasome pathway Liu et al. 2000;He et al. 2003). In constant darkness, FRQ is not only robustly rhythmic in its cellular concentration, but also in its phosphorylation states, so that the level and the phosphorylation status of FRQ define the time of the clock during a circadian cycle .Phoshorylation of FRQ is mediated by CKI, CKII, and a calcium/calmodulin-dependent kinase (Gorl et al. 2001;Yang et al. 2001Yang et al. , 2002Yang et al. , 2003. Molecular, genetic, and biochemical experiments have shown that phosphorylation of FRQ has several functions. Mutations of the FRQ phosphorylation sites lead to the stabilization of the FRQ protein and long period rhythms of the clock (Li...

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Section: Resultsmentioning

confidence: 99%

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Yang¹,

He²,

Cheng³

et al. 2004

Genes Dev.

111

117

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“…RIP acts during the haploid dikaryotic stage of the Neurospora sexual reproductive cycle, causing numerous C †G to T †A mutations within duplicated sequences. In a single passage through the sexual cycle, up to 30% of the C †G pairs in duplicated sequences can be mutated, with a strong preference for C to T mutations occurring at CpA dinucleotides 14 . The pattern of mutations produces a characteristic skewing of dinucleotide frequencies that allows RIPmutated sequences to be detected accurately 15 .…”

Section: Repeat-induced Point Mutationmentioning

confidence: 99%

The genome sequence of the filamentous fungus Neurospora crassa

Galagan

Calvo

Borkovich

et al. 2003

Nature

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1,498

1,226

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Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes

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“…Early genetic studies showed that RIP senses duplicated sequences and subjects both sequences to numerous polarized transition mutations (G:C to A:T) in the stage of the life cycle between fertilization and nuclear fusion Selker and Garrett, 1988). Up to B30% of the G:C pairs of duplicated sequences are mutated in a single passage through the sexual cycle (Cambareri et al, 1989;Cambareri et al, 1991). Although the mechanism of RIP remains to be elucidated, one of Neurospora's two putative DNA methyltransferases (DMT), RIP defective (RID), is essential for the process (Table 1) .…”

Section: Dna Methylation and Repeat-induced Point Mutation In Neurosporamentioning

confidence: 99%