Chizu Ishii

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

SUMMARY We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Isolation and characterization of MMS-sensitive mutants of Neurosporacrassa

Abstract Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or γ-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.

A novel phenotype of an excision-repair mutant in Neurospora crassa: Mutagen sensitivity of the mus-18 mutant is specific to UV

SummaryA UV-sensitive mutant has been isolated from UV-mutagenized conidia of Neurospora crassa. The mutation responsible for the lesion was mapped in linkage group VL, proximal to the nucleolus organizer region. We designated the mutant mus-18. The sensitivity of the mus-18 mutant to UV-irradiation was not particularly high, being less than twice that of the wild-type strain. However, the frequency of mutations at the ad-3 loci induced by UV was extremely high even at low doses, under conditions where survival rates of mus-18 cells were almost identical to those of wild-type cells. Photoreactivation of UV damage was normal in the mus-18 mutant. Sensitivity to other mutagens, such as gamma rays, 4-nitroquinoline-1-oxide, N-methyl-N′-nitro-N-nitrosoguanidine, mitomycin C and methyl methanesulfonate, was similar to that of the wild type. Fertility of the mus-18 mutant was normal in homozygous crosses. These results suggest that mus-18 is an excision-repair mutant. Measurement of endonuclease-sensitive sites (ESS) after liquid-holding recovery from UV damage revealed that ESS remained unrepaired for longer than 18 h in the mus-18 mutant, while most were eliminated within 6 h in wild-type cells and in other UV-sensitive mutants. This result suggests that mus-18 is defective in the incision step of dimer excision. The mus-18 mutant provides the first example of an excision-defective mutation in eukaryotes, which is specific to UV damage.

Validity of simplified scoring methods of somatic mutations in Tradescantia stamen hairs

Abstract In order to improve the time-consuming traditional scoring method of somatic mutations in Tradescantia stamen hairs, several simplified scoring methods were tested and compared for their statistical accuracy. For determining the number of pink mutant events per hair, it was found to be sufficient to count the number of hairs only on two oppositely located stamens, rather than counting those on all of the six stamens of each flower. For obtaining the number of pink mutant events per hair-cell division, it was also found to be sufficient to count the number of cells per hair (on 10 representative hairs each of two oppositely located stamens) for every two flowers, rather than for every flower. With these simplified scoring methods, it is possible to reduce the laboratory working time to about 60%, or to process about 1.7 times more samples during the same working time, resulting in little impairment to statistical reliability.

Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae

The mei-3 gene of Neurospora crassa encodes a homolog of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins, which are required for recombination and repair of DNA double-strand breaks. To determine the molecular function of MEI3 protein, anti-MEI3 antibody was prepared and used in Western blot analysis. The antibody cross-reacted only with crude extracts prepared from perithecia, the fruiting bodies of Neurospora. The molecular weight of the MEI3 protein was estimated to be 38 kDa. Transformation experiments showed that a DNA fragment longer than previously reported was needed to complement the mei-3 mutation. On sequencing cDNA and genomic DNA, one open reading frame (ORF) was found, which consists of three exons interrupted by two small introns. This ORF encoded a MEI3 protein of 353 amino acids, and the inferred MW of 38 kDa is in good agreement with the results from Western blot analysis. Comparisons of MEI3 with other Rad51 homologs indicated that MEI3 protein contains the two conserved core domains (I and II) generally observed in Rad51 homologs in eukaryotes. Northern blot analysis showed that expression of mei-3 was raised remarkably after UV-irradiation or methyl methanesulfonate (MMS)-treatment. The transcript size was 1.6 kb and this was also larger than was reported previously.

High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant.

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.

Isolation and genetic characterization of the Neurospora crassa REV1 and REV7 homologs: evidence for involvement in damage-induced mutagenesis

In a previous paper, we reported that the Neurospora crassa upr-1 gene is a homolog of the yeast gene REV3, which encodes the catalytic subunit of DNA polymerase zeta (polzeta). Characterization of the upr-1 mutant indicated that the UPR1 protein plays a role in DNA repair and mutagenesis. To help understand the mechanisms of mutagenic DNA repair in the N. crassa more extensively, we identified N. crassa homologs of yeast REV1 and REV7 and obtained mutants ncrev1 or ncrev7, which had similar phenotypes to the upr-1 mutant. Mutant carrying ncrev7 was more sensitive to UV and 4NQO, and slightly sensitive to MMS than the wild-type. The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild-type, but the sensitivity to 4NQO of the mutant was similar to that of the wild-type. In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, each of ncrev1 and ncrev7 mutants showed lower induced-mutability than the wild-type. Expression of ncrev1 and ncrev7 was found to be UV-inducible like the case of upr-1. Genetic analyses showed that the ncrev7 was identical to mus-26, which belongs to the upr-1 epistasis group, and that the ncrev1 was a newly identified DNA repair gene and designated as mus-42. Interestingly, all three mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype, not completely defective but inefficient in photoreactivation. These results suggest that N. crassa REV homolog genes function in DNA repair and UV mutagenesis through the bypass of (6-4) photoproducts.

A new UV-sensitive mutant that suggests a second excision repair pathway in Neurospora crassa

In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes. The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation. DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs). UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant. This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs. Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type. To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease. CPD excision in the mus-18 null mutant was severely affected but not completely inhibited. The double mutant showed a complete loss of the excision activity and was super sensitive to UV. These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway. The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA. Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway.

CLONING AND CHARACTERIZATION OF THE YEAST RAD1 HOMOLOG GENE (MUS-38) FROM NEUROSPORA CRASSA : EVIDENCE FOR INVOLVEMENT IN NUCLEOTIDE EXCISION REPAIR

Abstract A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32–37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene.

ATM and ATR homologes of Neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity.

Genome integrity is maintained by many cellular mechanisms in eukaryotes. One such mechanism functions during the cell cycle and is known as the DNA damage checkpoint. In the filamentous fungus Neurospora crassa, mus-9 and mus-21 are homologes of two key factors of the mammalian DNA damage checkpoint, ATR and ATM, respectively. We previously showed that mus-9 and mus-21 mutants are sensitive to DNA damage and that each mutant shows a characteristic growth defect: conidia from the mus-9 mutant have reduced viability and the mus-21 mutant exhibits slow hyphal growth. However, the relationship between these two genes has not been determined because strains carrying both mus-9 and mus-21 mutations could not be obtained. To facilitate analysis of a strain deficient in both mus-9 and mus-21, we introduced a specific mutation to the kinase domain of MUS-9 to generate a temperature-sensitive mus-9 allele (mus-9(ts)) which shows increased mutagen sensitivity at 37 degrees C. Then we crossed this strain with a mus-21 mutant to obtain a mus-9(ts) mus-21 double mutant. Growth of the mus-9(ts) mus-21 double mutant did not progress at the restrictive temperature (37 degrees C). Even at the permissive temperature (25 degrees C), this strain exhibited a higher mutagen sensitivity than that of the mus-9 and mus-21 single mutants, as well as slow hyphal growth and low viability of conidia. These results indicate that the mus-9(ts) mutation causes hypomorphic phenotypes in the mus-21 mutant and that these two genes regulate different pathways. Interestingly, we observed accumulation of micronuclei in the conidia of this double mutant, and such micronuclei were likely to correlate with spontaneous DSBs. Our results suggest that both mus-9 and mus-21 pathways are involved in DNA damage response, normal growth and maintenance of chromosome integrity, and that at least one of the pathways must be functional for survival.