Vacuum Ultraviolet Studies on the Nature of the Radiation Inactivation of Trypsin

Trypsin, in powder form and in frozen D(2)O-glucose solutions, at temperatures from 100 degrees to 300 degrees K, was excited with vacuum ultraviolet and near ultraviolet radiation to determine how absorbed photon energy is partitioned into radiative, nonradiative and/or inactivating processes; at 300 degrees K most of the absorbed energy is not reemitted, so that it (0.98-0.99 for excitation at 120 nm and 0.75-0.90 at 280 nm) is potentially available for inactivation. Since the effects of excitation wavelength and temperature on the emission quenching yields are generally different from those on the inactivation yields of dry trypsin, the mere retention of quenched energy by an enzyme does not necessarily lead to its inactivation. Thus, as predicted previously, the radiation inactivation of trypsin must proceed by rather specific mechanisms which undoubtedly depend upon environment-sensitive processes, since the nature of the molecular environment can modify the partitioning of energy so significantly; for example, there are differences in the phosphorescence-to-fluorescence ratio, in the activation energy for quenching, and in the lifetimes and kinetics of the decay of phosphorescence when trypsin in frozen glasses and dry trypsin are excited by various wavelengths of ultraviolet radiation.     

Selection of Autographa californica nuclear polyhedrosis virus for resistance to inactivation by near ultraviolet,far ultraviolet,and thermal radiation

Experiments were performed to isolate strains of Autographa californica nuclear polyhedrosis virus (Baculovirus) with inherent resistance to inactivation by far ultraviolet radiation, near ultraviolet radiation, and thermal radiation. Virus with apparently increased resistance to inactivation by near ultraviolet radiation was isolated. Virus with increased resistance to far ultraviolet radiation was not obtained. No significant differences in response to thermal radiation were observed between wild virus and virus selected for increased resistance to inactivation by this agent. Repeated selection treatment with far ultraviolet radiation and with near ultraviolet radiation resulted in the production of virus with significantly reduced virulence in comparison with wild virus. The virulence of heat-selected virus did not differ from wild virus.     

Differential effects of growth temperatures on inactivation and mutation of Candida albicans by ultraviolet radiation

Summary Candida albicans exhibits greater susceptibility to inactivation by ultraviolet (uv) radiation if grown before or after irradiation at 37° C rather than 25° C. Caffeine, acriflavin or amino acid analogues potentiate inactivation during postirradiation growth at 37° C but have little effect at 25° C. In contrast to inactivation, mutation induction by uv is unaffected by pre- or postirradiation growth temperatures or by metabolic antagonists. These findings are not explicable in terms of possible effects of growth temperatures on known mechanisms for repair of uv damaged DNA. They are consistent, however, with a previous proposal that a temperature dependent mechanism for dark recovery exists in C. albicans which involves synthesis of protein essential for repair of lethal, non-genetic uv damage.     

Relative susceptibilities of caffeine-sensitive and caffeine-resistant strains of Candida albicans to inactivation and mutation by ultraviolet radiation

Summary Stable variants having increased resistance to growth inhibition by caffeine were obtained from four different absolute, amino acid auxotrophs of Candida albicans. Differences in growth rates and expression of auxotrophy between the resistant (Caf^R) variants and their sensitive (Caf^S) progenitors suggest that caffeine resistance arises through suppressor mutations which affect the fidelity of messenger RNA translation.Both Caf^S and Caf^R strains of C. albicans are more susceptible to inactivation by ultraviolet radiation (uv) when grown at 37°C rather than 25°C following exposure. Post irradiation growth on caffeine potentiates ultraviolet inactivation of all Caf^S strains at both temperatures. Depending on its origin, a Caf^R strain (i) may show greater, lesser or the same intrinsic susceptibility to uv inactivation as its Caf^S parent at 25°C or at 37°C and (ii) may or may not be refractory to post-irradiation contact with caffeine. Caf^R variants independently isolated from a given auxotroph are alike in inactivational responses whereas those obtained from different auxotrophs are dissimilar. This implies that different suppressor mutations are unique in the way they affect expression of potentially lethal uv damage and that only one kind of suppressor is obtained by selection for caffeine resistance in a particular auxotroph.The histidine requiring Caf^R strain WB-2CR is much more resistant to uv inactivation that its Caf^S parent WB-2. Moreover, post-irradiation survival of WB-2CR is unaffected by caffeine. However, WB-2CR and WB-2 are equally susceptible to uv-induced reversion to prototrophy. In both strains, caffeine does not enhance uv-induced reversion at 25°C or 37°C and exhibits an antimutagenic activity at high uv dosage at 37°C.The findings reinforce previously reported indications that, in C. albicans, (i) caffeine-sensitive excision-repair of uv damaged DNA does not occur and (ii) caffeine potentiates uv cellular inactivation by disturbing post-irradiation synthesis of protein essential for recovery from non-genetic damage.     

Influences of cellular susceptibility to amphotericin B and of post-irradiation growth conditions on inactivation of Candida albicans by ultraviolet radiation

Earlier investigations have shown that inactivation of non-budding cells ofC. albicans by ultraviolet radiation is determined in part by (i) their post-irradiation incubation temperature, (ii) opportunity for exposure to long chain fatty acids or sterols and (iii) the nutri tional quality of their post-irraditation growth medium. These cultural conditions do not affect the viability of cells which are budding when irradiated nor the frequency of mutants among the survivors of irradiated, unbudded populations. Several lines of evidence have indicated that the post-irradiation growth conditions influence recoveries from non-genetic forms of uv-induced damage which are potentially lethal only for cells which are not in the process of division when irradiated. Studies reported here show that mutation to resistance to the polyene antibiotic, amphotericin B, changes the composite pattern of survival responses of uv treated unbudded cells to the three post-irradiation conditions. Acquisition of amphotericin B resistance does not alter the uv mutability of such cells nor the vulnerability of budding cells to uv inactivation. Differences in susceptibilities of cells to polyene antibiotics are known to be determined by differences in the composition and structural arrangement of their membranes. The present observations are consonant, therefore, with a previous proposal that post-irradiation temperature, nutrition and contact with lipid affect cellular recoveries from damages to processes which (i) are essential for initiation of cell division and (ii) are related to the organization of the cell membrane.     

Clastogenic effects of ftorafur. II. Cytogenetic analyses of bone marrow in mice

Our previous work has demonstrated that whereas near-UV radiation is not a mutagen for Haemophilus influenzae cells, it does induce mutations in purified transforming DNA. In order to test various hypotheses concerning this difference, we have irradiated cells at 334 and 365 nm, then lysed them and assayed the DNA for induced mutations and for inactivation of transforming ability. The inactivation was only a little lower than observed with highly purified transforming DNA. The DNA irradiated in vivo was mutated at both wave-lengths, but with considerably lower efficiency than was purified DNA. Neither incubation of the cells after irradiation and before lysis nor freezing and thawing the cells significantly changed the amount of mutation. It is concluded that there is some protection of the DNA against premutational lesions by the in vivo environment, but that it is not enough to account for the total lack of mutation of the cells. A probable explanation of this lack of cell mutation is that lethal lesions in the cells are induced much more readily than premutational lesions.     

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.     

An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation

Using dioleoylphosphatidylcholine liposomes incorporating various fatty acids and neutral lipids, we have examined the ability of such lipids to provide protection of Escherichia coli and vesicular stomatitis virus (VSV) against the lethal effect of ultraviolet (254 nm) radiation. While the presence of varying amounts of saturated (palmitic) or polyunsaturated (arachidonic) fatty acids or the lipid antioxidant, alpha-tocopherol, had little effect on killing by ultraviolet radiation, considerable radioprotection was observed with beta-carotene, retinal and vitamin K-1 at final concentrations of 1 mg/ml. In another approach, vesicular stomatitis virus grown under conditions in which its envelope fatty acid composition was substantially modified, showed little change in its sensitivity to inactivation by ultraviolet radiation. The results provide strong evidence for a radioprotective role of certain, relatively rare natural lipid components with conjugated polyene systems, but not of the more ubiquitous and abundant membrane fatty acids.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Isolation and Characterization of Conditional Alleles of Bacteriophage T4 Genes uvsX and uvsY

The bacteriophage T4 uvsW, uvsX and uvsY gene functions are required for wild-type levels of recombination and for normal survival and mutagenesis after treatments with ultraviolet (UV) and ionizing radiations. The ability of uvsX and uvsY mutations to suppress the lethality of gene 49 mutations was used to select temperature-sensitive and amber alleles of these two genes. (uvsW mutations do not suppress gene 49 mutations.) A simple and powerful complementation test was developed to assist in assigning uvs mutations to genes. The amber alleles of uvsX and uvsY behave as simple null alleles, fully suppressing a gene 49 defect, enhancing UV killing and abolishing UV mutagenesis. However, the properties of the ts alleles of uvsX and uvsY demonstrated that suppression of a gene 49 defect, sensitivity to UV-induced inactivation and UV mutability can be partially uncoupled. These results prompt the hypothesis that radiation mutagenesis occurs during DNA chain elongation past template damage within a recombinational intermediate rather than within a conventional replication fork.     

Radiation-induced dominant lethal mutations in oocytes of Musca domestica

Dominant lethal mutations induced by γ-radiation were measured in stage-7 and stage-14 oocytes of Musca domestica. At both stages the data are consistent with the multi-hit theory on radiation induction of dominant lethals. This conclusion is supported by fractionation experiments which indicate that both] S7 and S14 oocytes are capable of repairing, in defferent periods of time, a similar amount of dominant lethal damage.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Ultraviolet-induced alterations of the sodium inactivation in myelinated nerve fibres.

Ultraviolet radiation irreversibly reduces the sodium permeability in nerve membranes and, in addition, induces a change of the potential dependence of the kinetic parameters of sodium inactivation in the node of Ranvier. This second ultraviolet effect shifts the kinetic parameters of sodium inactivation h infinity (V), alpha h (V), and beta h (V) to more negative potentials (no changes of the slopes of the curves). The amount of the displacement delta V along the potential axis is equal for the three parameters and depends on the ultraviolet dose. It is about delta V = --10 mV after an irradiation dose of 0.7 Ws/cm2 at 280 nm. Both ultraviolet-induced effects depend on membrane potential and on the wavelength of the applied radiation. But while the potential shift is enhanced at more negative holding potentials, the ultraviolet blocking is diminished and vice versa. Further, the ultraviolet-induced potential shift is greater at 260 nm than at 280 nm, whereas a maximum sensitivity of ultraviolet blocking is found at 280 nm. Therefore, the two radiation effects are the result of two separate photoreactions. For explanation of the radiation-induced potential shift it is assumed that ultraviolet radiation decreases the density of negative charges at the inner surface of the nodal membrane. From this hypothesis a value for the inner surface potential psii was derived. --19 mV less than or equal to psii less than or equal to --14 mV.     

Shortvein, a New Component of the Decapentaplegic Gene Complex in DROSOPHILA MELANOGASTER

Our laboratory has been concerned with the structure and function of the decapentaplegic gene complex (DPP-C) in Drosophila melanogaster . To define the boundaries of the complex, we have studied the genetics of mutations allelic to a previously discovered mutation shortvein (shv ), known to reside near decapentaplegic. We found that shortvein resides distal to Hin-d and dpp within the same polytene chromosome doublet, 22F1-2. Lesions in shv can affect not only the formation of the wing veins but also can interfere with normal development of parts of the adult and/or be lethal. Like those of dpp mutants, the shv-associated adult abnormalities affect distal epidermal structures. Some shv lesions cause a larval lethal syndrome which is associated with an unusually long larval stage (ca. five to six times its normal duration). Lesions in shv exhibit an involved pattern of complementation with dpp mutations, indicating that both shv and dpp are parts of a single gene complex. A subset of the array of mutant phenotypes displayed by shv/dpp trans-heterozygotes appear to be dpp-specific phenotypes; we interpret these as reflecting an inactivation effect of certain shv alleles on dpp functions. The other abnormalities displayed by these trans-heterozygotes appear to be shv-specific defects; we view these as indicating an inactivation effect of certain dpp mutations on shv functions. Furthermore, embryonic lethal (EL) mutations within the DPP-C exhibit allelic interactions with all shv mutations. We conclude that the shortvein region represents a newly identified integrated portion of the DPP-C.     

Hydroxylamine-induced purple adenine (ad-3) mutants in Neurospora crassa. I. Characterization of mutants by genetic tests

The genetic effects of hydroxylamine (HA) on Neurospora crassa were studied in an effort to understand the difference between the results obtained on very simple prokaryotic systems and those obtained with mammalian systems. A 2-component heterokaryon was used to study the inactivation of conidia and the induction of recessive lethal mutations at specific loci and over the entire genome. The heterokaryon is heterozygous for 2 closely linked loci, ad-₃A and ad-₃B, in the ad-₃ region. Specific locus mutations can result from either point mutation or chromosome deletion. The results were as follows: (1) Both homokaryotic and heterokaryotic conidia had multi-hit survival curves, and there was no difference between the survival levels of the two as a function of treatment time. (2) The frequency of recessive lethal mutations in the ad-₃ region increased as the square of treatment time.     

Multiple, independent components of ultraviolet radiation mutagenesis in Escherichia coli K-12 uvrB5.

Reversion systems involving the lacZ53(amber) and leuB19)missense) mutations were developed to study the mutant frequency response of Escherichia coli K-12 uvrB5 (SR250) to ultraviolet radiation (254 nm). A one-hit mutant frequency response was discernible at ultraviolet radiation fluences below approximately 0.5 J m-2. At higher fluences the overall mutant frequency response could be resolved into one-hit and two-hit components. A new interpretation of the published data on E. coli K-12 indicates that SR250 is not unique in this respect. In addition, the Lac reversion system showed enhanced mutagenesis after ultraviolet radiation fluences of approximately 1 to 3 J m-2, whereas the Leu reversion system did not. We conclude that the complex ultraviolet radiation mutant frequency response curves for E. coli K-12 uvrB5 were the result of three independent mutagenic processes for Lac reversion and two for Leu reversion.     

Interactions Between Light and Gas Vacuoles in Halobacterium salinarium Strain 5: Effect of Ultraviolet Light

The potential light shielding by intracellular gas vacuoles in Halobacterium salinarium strain 5 was examined by looking at the ultraviolet light inactivation curves of both wild-type cells and mutants which are defective in the production of gas vacuoles. Whereas strains defective in gas vacuole production were slightly more sensitive to ultraviolet inactivation, no significant differences in ultraviolet sensitivity were seen, indicating that these subcellular inclusion bodies are not effective as light-shielding organelles. In addition, it was shown that ultraviolet light acts as a plasmid-curing agent in Halobacterium.     

Ultraviolet mutagenesis of radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans

A mutational tester strain (JP10) of the nematode C. elegans was used to capture recessive lethal mutations in a balanced 300 essential gene autosomal region. The probability of converting a radiation interaction into a lethal mutation was measured in young gravid adults after exposure to fluences of 254-nm ultraviolet radiation (UV) ranging from 0 to 300 Jm-2. Mutation frequencies as high as 5% were observed. In addition, three different radiation-hypersensitive mutations, rad-1, rad-3 and rad-7 were incorporated into the JP10 background genotype, which allowed us to measure mutation frequencies in radiation-sensitive animals. The strain homozygous for rad-3 was hypermutable to UV while strains homozygous for rad-1 and rad-7 were hypomutable. Data showing the effects of UV on larval development and fertility for the rad mutants is also shown and compared for wild-type and JP10 backgrounds.