Gamma-ray inactivation of conidia from heterokaryons of Neurospora crassa containing UV-sensitive mutations.

Two-component heterokaryons were formed with the fungus Neurospora crassa. The UV-sensitive mutations upr-I, uvs-4, and uvs-6 were utilized. Conidia produced by these heterokaryons were exposed to gamma-rays and survival curves were established for the three conidial fractions produced by each heterokaryon. Results showed that upr-I, when included in only one nuclear component, did not affect the sensitivity of any conidial fraction; however, when included in both components, all three conidial fractions exhibited two- to four-fold decreases in survival at the 30 krad exposure. The uvs-4 mutation, when included in one or both components, did not increase the sensitivity of any conidial fraction and appeared, in contrast, to impart a small increase in resistance to inactivation by gamma-rays. When included in only one component, uvs-6 increased the sensitivity of homokaryotic uvs-6 conidia but had no affect on the other two conidial fractions. When included in both components, uvs-6 resulted in exponential inactivation curves and at the 30 krad exposure, 100-fold decreases in survival for all three conidial fractions.     

A human enzyme that liberates uracil from DNA

An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).     

Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.     

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Conserved mRNA from the conidia of Neurospora crassa

Summary Species of RNA showing the characteristics of mRNA have been isolated from ungerminated conidia and from mycelia of Neurospora crassa grown for 8, 16 and 24 hours. Molecular hybridization between such RNA species and DNA together with hybridization competition between mRNA from ungerminated conidia and from growth periods of 8, 16 and 24 hours, showed that conidia contain conserved mRNA. Such mRNA may participate in protein synthesis taking place up to 30 minutes of incubation of the conidia.     

Repair replication in heterokaryons deprived from different repair-deficient xeroderma pigmentosum strains

Repair replication was studied in UV-irradiated cell populations obtained after fusion of cell strains originating from different xeroderma pigmentosum (XP) patients. The capacity to perform repair replication appeared to be restored completely in multinucleate heterokaryons resulting from fusion between a classic XP-strain and a De Sanctis-Cacchione (DSC) strain. In cell populations obtained by fusion of either two different classic XP strains or two different DSC strains no repair replication was observed.These results, obtained with the technique of density labelling and isopycnic centrifugation of DNA, confirm our previously reported results of autoradiographic studies of unscheduled DNA synthesis. The occurrence of complementation between a classic XP strain and a DSC strain indicates that the defect in the two forms of the disease is caused by different mutations.     

Three complementation groups in Cockayne syndrome

After 16 Jm-2 of UV-irradiation non-dividing normal cells recover normal rates of RNA synthesis within 24 h, whereas in cells from donors with Cockayne syndrome (CS) the rate of RNA synthesis gradually declines. Cultures of a mixed population from 2 CS donors were fused with polyethylene glycol; subsequently they were UV-irradiated and RNA synthesis was measured autoradiographically in mono-, bi-, and multinuclear cells. Genetic complementation was indicated by high levels of RNA synthesis in bi- and multinuclear cells when compared with mononuclear cells. Using this assay, 11 CS strains have been assigned to three complementation groups: 2 into group A, 8 into group B and 1 into group C. The strain in group C is derived from an individual who also had xeroderma pigmentosum (XP), and was the sole known representative of XP-complementation group B.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Thioguanine resistance, ouabain resistance and sister chromatid exchanges in V79/AP4 Chinese hamster cells treated with potassium dichromate

The biological activity of potassium dichromate (K2Cr2O7) was assayed in V79/AP4 Chinese hamster cells by measuring two mutational end points, thioguanine (TG) resistance and ouabain (OUA) resistance, and two non-mutational end points, cytotoxicity and sister chromatid exchanges (SCE). By exposing the cells for 1 h to the chemical, all biological end points examined were affected by the treatment in a dose-dependent manner. Moreover the combined use of the two selective systems indicated that chromium induces base-pair substitutions in mammalian cells in culture.     

Glutamine metabolism in nitrogen-starved conidia of Neurospora crassa.

During nitrogen deprivation, de novo synthesis of glutamine synthetase was induced in non-growing conidia of Neurospora crassa. When ammonia or glutamine was added to conidia which had been deprived of nitrogen, glutamine and arginine accumulated at a higher rate than in condia not deprived of nitrogen. The degradation of exogenous glutamine to glutamate is apparently a necessary step in the accumulation of glutamine and arginine within the conidia. In non-growing conidia, a cycle probably operates in which glutamine is degraded and resynthesized. The advantages of such a cycle would be that the carbon and nitrogen could be used to synthesize amino acids in general, as well as for the synthesis and accumulation of arginine and/or glutamine in particular.     

Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.     

Excision repair by human fibroblasts of DNA damaged by r-7, t-8-dihyroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo(a)pyrene

Benzo(a)pyrene diol-epoxide I (r-7,t-8,dihydroxy-t-9,10 oxy-7,8,9,10 tetrahydrobenzo(a)pyrene) was used to treat either human adenovirus 5 or cultures of human fibroblasts. The survival of diol-epoxide I treated adenovirus was greater when infecting fibroblasts from normal persons than when infecting fibroblasts from patients with xeroderma pigmentosum (XP). One diol-epoxide I molecule bound per viral genome correlated with one lethal hit as measured using XP fibroblasts.

Normal fibroblasts blocked in semi-conservative DNA synthesis incorporated into their DNA more [³H]thymidine in response to diol-epoxide I treatment than did XP fibroblasts, and also excised more diol-epoxide I from their DNA. All of the effects described above were similar to those obtained when the inactivating agent was ultraviolet light rather than benzo(a)pyrene diol-epoxide I.     

A deficiency in the repair of UV and γ-ray damaged DNA in fibroblasts from Cockayne's syndrome

The host-cell reactivation of V antigen production for irradiated adenovirus was examined in fibroblasts from 5 unrelated patients with Cockayne's syndrome (CS) and 2 CS heterozygotes. The fibroblast cultures were infected with either irradiated or non-irradiated adenovirus and subsequently examined for the presence of viral structural antigens using immunofluorescent staining. All CS-homozygous strains showed a reduced host-cell reactivation (HCR) of this viral function for both UV- and γ-irradiated virus. For UV-irradiation of the virus, D₃₇ values expressed as a percentage of that obtained on normal strains, ranged from 14 to 35%. For γ-irradiation of the virus these values ranged from 61 to 80%. These results indicate some defect in the repair of both UV- and γ-ray-induced DNA damage for CS. 1 CS-heterozygote strain tested also showed a reduced HCR for UV-irradiated adenovirus intermediate between that of the patient strain and normal, whereas another CS-heterozygote strain showed an apparently normal HCR level.     

Nuclear division cycle in germinating conidia of Neurospora crassa.

A temperature-sensitive mutant has been shown to be blocked at a specific point in the nuclear division cycle: just before the initiation of DNA synthesis at the time when the spindle pole bodies have duplicated but not separated. The metabolic activities of conidia of this mutant strain at the nonpermissive temperature have led us to conclude that the nuclei in a population of dormant conidia are arrested at various points in the nuclear division cycle. This conclusion is substantiated by the activities of conidia in the presence of the inhibitory drugs cycloheximide and hydroxyurea. In each inhibitory situation we observed that some, but not all, of the conidia were able to accomplish DNA synthesis and/or nuclear division.     

An inducible acetate transport system in Neurospora crassa conidia.

Neurospora crassa conidia possess an active transport system for the uptake of acetate. This system was characterized as: (a) energy dependent; (b) taking place against a concentration gradient; (c) saturating at higher substrate concentrations and (d) competitively inhibited by propionate. Activity of the acetate transport system can be further enhanced by preincubating conidia in 1 mM acetate medium for 180 min (the inducible transport system). The conidial system and the inducible system have similar properties. The development of the inducible transport was dependent on RNA and protein synthesis. A genetic control of this system was further confirmed by isolating a mutant acp-i acetate permease, inducible) that fails to develop the inducible transport system.