Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans

Hooley, P.

Shawcross, S. G.

Strike, P.

English

Kansas State University

Fungal Genetics Reports Volume 34 Article 8 Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans. P. Hooley S. G. Shawcross P. Strike Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Hooley, P., S.G. Shawcross, and P. Strike (1987) "Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans.," Fungal Genetics Reports: Vol. 34, Article 8. https://doi.org/10.4148/1941-4765.1554 This Regular Paper is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans. Abstract Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol34/iss1/8 The binding of [^3 H]Gpp(NR)p and [^35 S]GTPYS to proteins was assessed for theircapacity to bind them in the presence of GTP. In addition to the standard reaction mix-ture, crude extract containing membrane components and various concentrations of GTP orATP were added, and the radioactivity retained on the filter was counted (Table 1).  GTPbut not ATP apparently inhibited the binding of [^3 H]Gpp(NH)p and [^35 S]GTPyS to proteinssupporting the idea that these proteins were GTP-binding proteins. The efficiencies ofbinding of [^3 H]Gpp(NH)p and of [^35 S]GTPYS to GTP-binding proteins were similar, indica-ting both compounds to be equally useful to assay GTP-binding proteins. We are gratefulto Mrs. M. Yazawa and Miss T. Imaizumi for excellent technical assistance.- - - National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki  444Hooley. P., S.G. Shawcross               Studies of DNA repair in Aspergillus nidulanshave resulted in the isolation of a number ofstrains showing increased sensitivity to a rangeof mutagenic treatments (e.g. Jansen  1970 Mut.Res. 10:21-32: Fortuin 1971 Mut. Res. 11:149-and P. StrikeRelationships between pyrimidine162).  Recently, the isolation of strainsmetabolism and sensitivity to sensitive to damage induced by alkylating agentsalkylating agents in(Swirski et al, manuscript submitted) has intro-duced the possibility of identifying particulareukaryotic  DNA repair enzymes. The development ofAspergillus nidulans a high frequency transformation vector (pDJB3)for A. nidulans (Ballance and Turner 1985 Gene36:321-331) now makes feasible the cloning of genes coding for such enzymes.  The plasmidpDJB3 relies on complementing a defect in pyrimidine metabolism of the "target" strain(G191) for recognition of transformants.  In Ustilago madis, some strains which aresensitive to radiation and alkylating agent damage can also exhibit deficiencies inpyrimidine metabolism (Moore 1975 Mut. Res. 28:355-366).  Consequently, the cloning ofgenes affecting DNA repair in A. nidulans using genomic DNA banks in pDJB3 may provealkylating agents.impractical if mutations affecting pyrimidine metabolism also affect response toWe report here a preliminary study of the effects of mutations at two loci (pyrE andpyrG) controlling pyrimidine biosynthesis (Palmer and Cove 1975 MGG 138:243-255) uponsensitivity to the alkylating agent MNNG (N-methyl-N-nitro-N-nitrosoguanidine).Initially, in growth tests on solid media containing MNNG, G191  (pyrG89 pabaA1 fwA1uaY9) was found to be slightly more sensitive than wild-type, and G190 (pyrE8 anA1 luA1yA2 cnxH5) very sensitive.  In order to test whether the mutations at the pyrE or pyrGloci affected response to alkylating agents, normal wild-type activity was restored in twoways.  Two transformants of G191 (pyrG) were selected following Ballance and Turners(1985) technique, whilst some nineteen revertants of G190 (pyrE) were selected followingUV irradiation.  Both the transformant class and the revertant class had functional pyrGand pyrE activity restored respectively and could grow in the absence of pyrimidinesupplementation.  Sexual progeny of one such pyrE revertant tested showed no segregationsuppressor system (Palmer and Cove, 1975).  When tested for MNNG response, both revertantof pyrE, confirming that true reversion had occurred at the pyrE locus and not in some(pyrE^+) and transformant (pyrG^+) classes showed sensitivities indistinguishable from theirparental (G190 and G191) strain responses in growth tests on solid media.  Thus theincreased sensitivity to MNNG of strains G190 and 6191 could not be the result ofmutations at either the pyrE or pyrG loci.A recombinant strain harboring pyrG and two mutations conferring sensitivity toalkylating agents, saA1 and sagC3 (Swirski et al, manuscript submitted) showed no changein MNNG response with change in the level of pyrimidine supplementation.Additionally, when a heterozygous dipoid between G190 and a strain containing sagC3was constructed, it showed a wild-type level of resistance to MNNG confirming that thesensitivity of G190 was not due to mutation of the sagC3 locus, although sagC3 mapsclosely to pyrE.In conclusion, genomic banks of pDJB3 may be used to screen for complementation ofmutations conferring sensitivity to alkylating agents, as pyrG has no detectable effectupon response to MNNG.  The MNNG sensitivity of G190, however, suggests that complex stockstrains may harbor mutations other than those for which they were selected.  While thepresent study has failed to show an interaction between pyrimidine metabolism andsensitivity to alkylating agents, the number of loci involved in both systems in A.nidulans (Palmer and Cove, 1975; Swirski et al, manuscript submitted) may still make suchrelationships of relevance to DNA repair in Aspergillus. - - - Dept. of Genetics,  University of Liverpool, P.O. Box 147, Liverpool L69 3BX, United KingdomM.S. Islam and A. Nessa We have been investigating the molecularmechanisms of cellular ageing using NeurosporaCell Biology of ageing. IV. Effect of  crassa as the test organism.  By subjecting theconidia to gamma radiation a number of earlyNordihydro-guaiaretic acid(NDGA)  and senescent mutants showing varied ageingsyndromes have been isolated and characterizedcortisone on the ageing syndromes of   (Islam et al. 1981 Cell Biol. Int. Rep. 5:1005-1017).  In general, these mutants show (i) de-early senescent mutants of N. crassa creased extensional growth, (ii) low replicationpotentials, (iii) less conidial  viability, (iv)less biomass formation (in some cases), (v) increased accumulation of 'Lipofuscin' - agepigment (in some cases), and (vi) increased malondialdehyde formation - a product oflipid peroxidation.  In an attempt to overcome the deleterious effects of 'free radicals'which were believed to be the primary cause of early senescence in these mutants, wepreviously studied the effects of some antioxidants (free radical scavengers) likevitamin E, vitamin C and sodium selenite (Islam and Nessa 1983 Cell Biol. Int. Rep.7:404.  More recently, the effects of NDGA and cortisone, which are also known to bepotent antioxidant and membrane stabilizer, respectively (Rana and Munkres 1978 Mach.Ageing and Dev. 7:241-272) were investigated. The results are shown in Table 1 and Table2. The results indicated that NDGA had a significant positive effect on 377 and corti-sone on 270 in the case of malondialdehyde formation i.e. less of the aldehyde was foundto accumulate in the supplemented cultures than in the controls (measured according toHeath and Packer 1968; see Islam and Nessa 1984 Cell Biol. Int. Rep. 8:373-377).  Forother mutants NDGA had either significantly negative or very little negative effect,while cortisone had very little positive effect.  In the case of other syndromes of earlysenescence, NDGA or cortisone had very little or no beneficial effect (Table 1 and 2).The results indicate that cortisone could be a more potent quencher of excessive lipidperoxidation than NDGA as all the mutants tested responded to some extent positively toit with respect to malondialdehyde formation. But, since for the other syndromes ofearly senescence cortisone had very little beneficial effect, its overall role in ageingcould not be proved unambiguously.Table 1.  Effect of Nordihydroguaiaretic acid (NDGA; 20uM) on Ageing mutants of N. crassa|strain |Malondialdehyde  |UV-fluorescence  of|       |formation after  |culture filtrate|      |72 hrs.|       |535nm-6OOnm)cont.377 NDGAcont.448 NDGAcont.EmA   NDGA0.89** + 0.010.96  +   0.100.04* + 0.030.1*  + 0.03Cont. 0.76* + 0.14  Light green270  NDGA 0.96* + 0.04  Light greenBiomass    Linear  |Growth       |Conidial    |produc-    growth  |potentia-    |viability   |tion after up to   |lities in    |after 15   |7 days    14th day|liquid me-   |days       ||       |Mean^a + S.D.    |                      (mg/lOml) (in cm) |dia (No. of  |(Survival   ||       |                 |                                          |subcultures  |percentage) ||       |                 |                                          |survived     |            |25.0        107.1     16         035.0        102.7     13         0.44No fluorescence 52.5         86.9      6         0No fluorescence 60.0         79.2      4         01.09** + 0.05 Light greenLight green0.81 + 0.16  Very lt. greenVery lt. green45.0 23.3      4         042.5 21.3      4         050.0 32.5     13         2.050.0 35.4     13         1.1No fluorescenceNo fluorescence45.0 132.5    >18       10053.75       129.5     13       100a: Average of two independent experiments*: Significant at 5% level **: Significant at 1% level

Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans.

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Relationships between pyrimidine metabolism and sensitivity to alkylating agents in Aspergillus nidulans.

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