The Vacuolar ATPase of Neurospora crassa Is Indispensable: Inactivation of the vma-1 Gene by Repeat-Induced Point Mutation

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 147-154 ◽  
Author(s):  
Tracy L Ferea ◽  
Barry J Bowman
Keyword(s):  
Neurospora Crassa ◽  
Point Mutation ◽  
Point Mutations ◽  
Vacuolar Atpase ◽  
Catalytic Subunit ◽  
Multiple Point ◽  
Extra Copy ◽  
Process Sequencing ◽  
Functional Copy ◽  
Repeat Induced Point Mutation

Abstract To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

scholarly journals Synthesis of Signals for De Novo DNA Methylation in Neurospora crassa

2003 ◽  
Vol 23 (7) ◽  
pp. 2379-2394 ◽  
Author(s):  
Hisashi Tamaru ◽  
Eric U. Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
Point Mutation ◽  
Dna Sequences ◽  
De Novo ◽  
Test Site ◽  
Minor Groove ◽  
Binding Motif ◽  
Repeat Induced Point Mutation

ABSTRACT Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA) n or (TTAA) n were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5′ of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.

RIP (repeat induced point mutation) as a tool in the analysis of P-450 and sterol biosynthesis in Neurospora crassa

1991 ◽  
Vol 19 (3) ◽  
pp. 799-802 ◽  
Author(s):  
Ian F. Connerton ◽  
Shelly M. Deane ◽  
Jenny A. Butters ◽  
R. S. Thomas Loeffler ◽  
Derek W. Hollomon
Keyword(s):  
Neurospora Crassa ◽  
Point Mutation ◽  
Sterol Biosynthesis ◽  
Repeat Induced Point Mutation

scholarly journals The Neurospora crassa standard Oak Ridge background exhibits an atypically efficient meiotic silencing by unpaired DNA

2018 ◽  
Author(s):  
Dev Ashish Giri ◽  
Ajith V. Pankajam ◽  
Koodali T. Nishant ◽  
Durgadas P. Kasbekar
Keyword(s):  
Genetic Variation ◽  
Neurospora Crassa ◽  
Point Mutation ◽  
Genetic Background ◽  
Meiotic Silencing ◽  
Genetic Studies ◽  
Oak Ridge ◽  
General Abstract ◽  
Repeat Induced Point Mutation ◽  
Made In

AbstractMeiotic silencing by unpaired DNA (MSUD) was discovered in crosses made in the standard Oak Ridge (OR) genetic background of Neurospora crassa. However, MSUD often was decidedly less efficient when the OR-derived MSUD tester strains were crossed with wild-isolated strains (W), which suggested either that sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was much less efficient in near-isogenic crosses made in a novel N. crassa B/S1 and the N. tetrasperma 85 genetic backgrounds. Possibly, regulatory cues that in other genetic backgrounds calibrate the MSUD response are missing from OR. The OR versus B/S1 difference appears to be determined by loci on chromosomes 1, 2, and 5. OR crosses heterozygous for a duplicated chromosome segment (Dp) have for long been known to exhibit an MSUD-dependent barren phenotype. However, inefficient MSUD in N. tetrasperma 85 made Dp-heterozygous crosses non-barren. This is germane to our earlier demonstration that Dps can act as dominant suppressors of repeat-induced point mutation (RIP). Occasionally, during ascospore partitioning rare asci contained >8 nuclei, and round ascospores dispersed less efficiently than spindle-shaped ones.General abstractIn crosses made in the standard OR genetic background of Neurospora crassa, an RNAi-mediated process called MSUD efficiently silences any gene not properly paired with its homologue during meiosis. We found that MSUD was not as efficient in comparable crosses made in the N. crassa B/S1 and N. tetrasperma 85 backgrounds, suggesting that efficient MSUD is not necessarily the norm in Neurospora. Indeed, using OR strains for genetic studies probably fortuitously facilitated the discovery of MSUD and its suppressors. As few as three unlinked loci appear to underlie the OR versus B/S1 difference in MSUD.

scholarly journals Repeat-Induced Point Mutation (RIP) as an Alternative Mechanism of Evolution Toward Virulence in Leptosphaeria maculans

2009 ◽  
Vol 22 (8) ◽  
pp. 932-941 ◽  
Author(s):  
Isabelle Fudal ◽  
Simon Ross ◽  
Hortense Brun ◽  
Anne-Laure Besnard ◽  
Magali Ermel ◽  
...  
Keyword(s):  
Point Mutation ◽  
Leptosphaeria Maculans ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Avirulence Genes ◽  
Frequent Mutation ◽  
Stop Codons ◽  
Single Field ◽  
First Time ◽  
Repeat Induced Point Mutation

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.

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