Alice L. Schroeder

ULTRAVIOLET-SENSITIVE MUTANTS OF NEUROSPORA. II. RADIATION STUDIES.

SummaryTwo new, recessive, chromosomal mutants of Neurospora, uvs-3, and uvs-4, have been found to be more sensitive to UV than wild type. The uvs-3 mutant, is very sensitive to nitrogen mustard, nitrosoguanidine and X-rays. UV-dose-response curves of uvs-3 lack the shoulder typical of wild type but parallel the wild-type curve at low survival levels. Unlike wild type, which shows a decrease in survival when incubated in water or proflavine (4 mg/ml) after UV, uvs-3 shows an increase in survival after water incubation and an even greater increase when incubated in proflavine. It also differs from wild type in showing much less photoreactivation and in not losing its photoreactivation ability during two hours incubation in water.The uvs-4 mutant is not sensitive to nitrogen mustard and only slightly sensitive to nitrosoguanidine. Dose-response curves of uvs-4 retain a shoulder and are steeper than those of wild type at low survival levels. Proflavine sensitivity and photoreactivation of uvs-4 resemble wild type, but no decrease in survival occurs when it is held in water after UV.The combination of characteristics shon, by uvs-4 is not like that of any of the UV-sensitive mutants found in E. coli but those of uvs-3 resemble those shown by the EXR (X-ray sensitive, low to normal recombination) and reckless REC (X-ray sensitive, recombinationless) mutant classes in bacteria.

Chromosome instability in mutagen sensitive mutants of Neurospora

SummaryFour mutagen sensitive mutants of Neurospora (mus-7, mus-9, mus-11, and mei-2) are shown to increase mitotic chromosome instability in the duplication test developed by Newmeyer. Three other mutagen-sensitive mutants (upr-1, mus-8, and mus-10) do not increase chromosome instability. Previously three mutagen-sensitive mutants (uvs-3, uvs-6, and mei-3) were also shown to increase chromosome instability. The growth of all seven mutants that increase chromosome instability, is shown here to be more sensitive to hydroxyurea than that of wild type. Hydroxyurea, a compound which inhibits the enzyme ribonucleotide diphosphate reductase, is also shown to increase chromosome instability in the absence of any mutagen-sensitive mutation. These seven mutations are known to represent seven different genes in two epistasis groups. They have been shown previously to have four other properties in common: meiotic defects and sensitivity to y-rays, methyl methane sulfonate and the amino acid histidine. Their shared properties lead to the prediction here that all have reduced or altered deoxyribonucleotide pools.

Properties of a UV-sensitive neurospora strain defective in pyrimidine dimer excision

Abstract The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains. Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene. These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.

Deoxyribunucleoside triphosphate pools in Neurospora crassa: effects of histadine and hydroxyurea

Abstract An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crass has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/μg DNA for dGTP to 24.2 pmoles/μg DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ration of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other poolds remains the same. In the presence of 11 mM histadine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with free radical at the active sites, as does hydroxyurea.

γ-Ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telagiectasia cell lines

Abstract Well characterized γ-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telagiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characterteristics of γ-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Norman human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline gradient centrifugation, repression of DNA synthesis by low doses of γ-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.

mei-2, a mutagen-sensitive mutant of Neurospora defective in chromosome pairing and meiotic recombination

SummaryA Neurospora crassa mutation, mei-2, affecting meiosis and mutagen sensitivity, was characterized for its effect on meiotic recombination and chromosome pairing. Results from homozygous mei-2 crosses involving distant markers on the same chromosome demonstrated a drastic reduction in meiotic recombination. However, mitotic recombination continued to occur. Cytological observations indicated that pairing of homologous chromosomes in zygotene was greatly reduced or absent, resulting in aberrant segregation at anaphase I and often at subsequent divisions as well. The few mature ascospores produced were frequently disomic for one or more chromosomes.

Postreplication repair in Neurospora crassa

SummaryChanges in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.

Homologous recombination following transformation in Neurospora crassa wild type and mutagen sensitive strains

In filamentous fungi transformed with linear DNA, the frequency of gene replacement varies from a few percent in Neurospora crassa, where it depends on the extent of homology between the transforming DNA and the gene to be replaced, (Asch and Kinsey 1990 Mol. Gen. Genet. 22:37-43) to about 30% in Aspergillus (Miller et al. 1985 Mol. Cell Biol. 5:17-14). Yet, in the yeast, Saccharomyces cerevisiae, homologous gene replacement is the rule and ectopic integration of transforming DNA is extremely rare (Fincham 1989 Microbiol. Rev. 53:148-170). This variation could be the result of the action of one or a few genetic pathways and thus, be affected by single mutations. Mutants with altered ability to integrate transforming DNA may be sensitive to ionizing radiation, since double strand break repair is necessary for both recombination and survival after ionizing radiation damage. Therefore, we looked among the radiation-sensitive mutants, uvs-2,3,6, mus-9,11, and mei-2,3, for changes in the ability to integrate transforming DNA either ectopically or by homologous recombination. All mutant strains were extensively backcrossed to wild type strains of 74-OR23-1A background and the 74-OR23-1A strain served as a control. Authors Alice L. Schroeder, Martin L. Pall, James Lotzgesell, and Jacie Siino This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol42/iss1/20 Homologous recombination following transformation in Neurospora crassa wild type and mutagen sensitive strains Alice L. Schroeder, Martin L. Pall, James Lotzgesell and Jacie Siino, Dept of Genetics and Cell Biology, Washington State University, Pullman, WA 99164-4234. In filamentous fungi transformed with linear DNA, the frequency of gene replacement varies from a few percent in Neurospora crassa, where it depends on the extent of homology between the transforming DNA and the gene to be replaced, (Asch and Kinsey 1990 Mol. Gen. Genet. 22:37-43) to about 30% in Aspergillus (Miller et al. 1985 Mol. Cell Biol. 5:17-14). Yet, in the yeast, Saccharomyces cerevisiae, homologous gene replacement is the rule and ectopic integration of transforming DNA is extremely rare (Fincham 1989 Microbiol. Rev. 53:148-170). This variation could be the result of the action of one or a few genetic pathways and thus, be affected by single mutations. Mutants with altered ability to integrate transforming DNA may be sensitive to ionizing radiation, since double strand break repair is necessary for both recombination and survival after ionizing radiation damage. Therefore, we looked among the radiation-sensitive mutants, uvs-2,3,6, mus-9,11, and mei-2,3, for changes in the ability to integrate transforming DNA either ectopically or by homologous recombination. All mutant strains were extensively backcrossed to wild type strains of 74-OR23-1A background and the 74OR23-1A strain served as a control. A plasmid, pMTR::HYG (Figure 1), was designed to allow rapid distinction between transformants arising through homologous recombination events and those arrising through ectopic integration events. [Since we did not distinguish between homologous gene replacement and the other more complex changes that are occasionally found when exogenous DNA recombines with an endogenous homologous sequence (Miao et al. 1995 Genetics 139:15331544), these events will simply be called homologous events and ectopic events.] This plasmid carries the amp and ori segment and a 2.7 kb segment of Neurospora crassa DNA including the entire coding region of the mtr (methyl tryptophan resistance) gene. It was constructed by inserting a 2397 bp SalI fragment containing the hygromycin resistance gene (hyg or hph) plus the trpC promoter from pCSN44 (Staben et al. 1989 Fungal Genet. Newslet. 36:79-81) into a XhoI site within the mtr coding sequence of pCVN2.9 (Koo and Stuart 1991 Genome 34:644651). Hygromycin resistance is dominant, so all spheroplasts transformed with this construct will be hygromycin resistant (HYGr). The mtrphenotype is recessive. The mtr gene controls the uptake of neutral amino acids including the toxic amino acids, 4-methyl tryptophan (leading to the mtr designation) and p-fluorophenylalanine (FPA). Transformants with an intact endogenous mtr gene will be sensitive to these compounds while transformants, in which the endogenous gene has been replaced with , or disrupted by the exogenous, disrupted gene, will be resistant (FPAr). Spheroplasts were transformed as described by Vollmer and Yanofsky (1986 Proc. Natl. Acad. Sci. USA 83:4869-4873) with pMTR::HYG, cut with XhoI and SalI in the N. crassa DNA outside the mtr coding regions (see Figure 1). They were plated in top agar on sorbose minimal medium containing 200 μg/ml HYG. After 3 6 days, pieces of colonies which had grown sufficiently to break through the top agar and produce conidial hyphae were transferred to 0.5 ml liquid minimal medium containing 200 μg/ml HYG. After 4 to 8 days, conidia or hyphae from 1 Schroeder et al.: Homologous recombination following transformation in Neurospora c Published by New Prairie Press, 2017 these tubes were suspended in water and spotted on sorbose minimal medium with no drug, 200 μg/ml HYG or 15 μg/ml FPA, and scored for growth after 36-48 h at 30°C. Isolates which grew on FPA were further tested by spreading a dilute conidial suspension from the HYG tube on FPA containing plates. Transformants in which recombination has occurred at the homologous mtr gene will produce many FPA resistant colonies. Transformants in which DNA has integrated at ectopic sites will be FPA sensitive unless they have acquired a spontaneous mutation in the mtr gene while growing in liquid medium or on the plate. Such mutants will have few, if any, FPA resistant colonies, unless the mutation occurred very early. To show further that the transformants identified as due to events at the mtr locus were due to the presence of exogenous DNA at the mtr gene, crosses to a col-4mtr+ a strain were made. Single conidial isolates of the HYGr FPAr transformants were picked either from the FPA plates, or from HYG plates and shown to be HYGr FPAr. They were grown on minimal medium and then grown on minimal medium containing FPA to eliminate any FPAs nuclei before crossing. The col-4+ progeny from these crosses were scored for sensitivity to HYG and FPA. Because col-4 is only 1 map unit from mtr, the col-4+ progeny of such a cross should be hyg+ mtrwith a HYGr FPAr phenotype, except for rare col-4+ hygmtr+ (HYGs FPAs) recombinants. Transformants which have an ectopic copy of the transforming DNA and an independent mtrmutation should give col-4+ progeny with a ratio of the HYGr FPAr and HYGs FPAr phenotypes of 1:1. Both transient and stable transformants are produced when spheroplasts are transformed with pMTR::HYG. It was not possible to quantify the total number of transformants obtained. However, no large, i.e. >5x, difference in transformation frequency was observed between 74OR23-1A and the mutant strains. Of transformants which had grown sufficiently to break the agar surface (see Table I), greater than 90% proved stable for HYGr in all strains except mus-9 (80%) and uvs-6 (56%). We have observed a similar instability of benomyl resistance in uvs-6 conidia transformed with bml carrying plasmids. As shown in Table I, the mutagen sensitive strains appear to fall into two classes when the frequencies of homologous recombination events among total transforming events are compared. One group, mei-2, mus-9, and mus-11, has frequencies of homologous events similar to wild type. A second group, uvs-2, uvs-3, uvs-6, and mei-3, has lower frequencies of homologous events. In uvs-6 and mei-3, where sufficient isolates have been examined, this frequency is significantly lower than in 74-OR23-1A. Table I. Transformation of Neurospora by the pMTR::HYG plasmid. Strain # of HYGr HYGr FPAr Frequency of Homologous isolates transtransformants HYGr FPAr events formants transformants confirmed by crosses 74-OR23-1A 705 669 16 0.024 16/16 74-OR23-1A(a)200 200 4 0.02 1/4 mei-2 200 200 4 0.02 1/3 mus-9 250 201 3 0.015 0/1 mus-11 291 274 6 0.022 -uvs-2 300 297 2 0.0067(b) 0/2 uvs-3 250 233 1 0.0043(b) -uvs-6 1101 619 3 0.0048(c) 1/1 mei-3 550 527 3 0.0057(c) -2 Fungal Genetics Reports, Vol. 42 [1995], Art. 20 https://newprairiepress.org/fgr/vol42/iss1/20 DOI: 10.4148/1941-4765.1350 (a)The XhoI and SalI cut plasmid used in these transformations was blunt ended with the Klenow fragment of DNA polymerase I, dCTP, dTTP, dGTP and dideoxyATP. (b)Chi square; p<0.1. (c)These values are significantly different by chi square from transformation in 74-OR23-1A at p<0.01 When the 16 HYGr FPAr transformants in 74-OR23-1A were crossed to the col-4mtr+ strain, the col-4+ progeny were primarily the HYGr FPAr phenotype expected if these transformants had arisen through an homologous gene replacement or insertion event. About 1.5% of the col4+ progeny were HYGs FPAs recombinant progeny. Thus, all 16 of these transformants arose through recombination events at the mtr locus (rightmost column, Table I). A few (3 each) unexpected FPAr HYGs and FPAs HYGr progeny were seen. The FPAr HYGs progeny could be the result of the presence of an additional, ectopic insertion of the pMTR::HYG plasmid allowing RIP during the cross which would inactivate the hyg+ DNA but would have no effect on the FPAr phenotype, since the mtr gene is already inactive. When the HYGr FPAr isolates from the mutagen sensitive strains were crossed to col-4 mtr+, crosses from 7 such transformants produced ascospores. Two, a mei-2 isolate and a uvs-6 isolate, gave only HYGr FPAr progeny, indicating that they arose through homologous recombination events. A uvs-2 isolate gave 13 HYGr FPAr : 4 HYGs FPAr progeny and thus probably occurred via an ectopic event with a subsequent mutation in the mtr gene or had both homologous and ectopic integration of DNA followed by RIP of the hyg gene during th

Deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of Neurosporacrassa

Deoxyribonucleoside triphosphate (dNTP) levels were measured in wild type Neurospora and nine mutagen-sensitive mutants, at nine different genes. Eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dNTPs. Two patterns were seen. Five of the mutants had altered ratios of dNTPs, with relatively high levels of dATP and dGTP and low levels of dCTP, but changes in the dTTP/dCTP ratio did not correlate with changes in spontaneous mutation levels. During exponential growth all but two of the mutants had small but consistent increases in dNTP pools compared to wild type. DNA content per microgram dry hyphae was altered in several mutants but these changes showed no correlation with the dNTP pool alterations.

Ionizing irradiation effects on S-phase in checkpoint mutants of the yeast Saccharomyces cerevisiae

Abstract. In mammalian cells, γ-irradiation activates checkpoint controls to delay entry into, or passage through S-phase, while chronic exposure to methyl methanesulfonate or hydroxyurea causes a similar delay in yeast. In yeast, at least five genes are involved: RAD9, RAD17, RAD24, RAD53 and MEC1, a homologue of ATM. Here, using flow cytometry analysis and alkaline sucrose gradient centrifugation of labeled, newly made DNA, we demonstrate, in synchronized RAD wild-type Saccharomyces cerevisiae cells, that: (1) γ-irradiation at START delays entry into S-phase, (2) irradiation shortly before or during early S-phase delays completion of S-phase and (3) the latter response is largely a consequence of replicon initiation inhibition. The delay produced by irradiation during early S-phase depends on the function of the checkpoint genes RAD9, RAD17, RAD24, RAD53, MEC1 and MEC3. However, at least four, RAD17, RAD53, MEC1, MEC3, are not needed to delay S-phase progression when cells are irradiated shortly before S-phase begins.