Search

We present methods for the determination of UVA-induced binding of 8-methoxypsoralen (8-MOP) to nucleic acids and protein and for a quantitative assay of radioactively labelled 8-MOP plus UVA induced DNA photoproducts in the yeast Saccharomyces cerevisiae . For the dose range up to 60 kJ M −2 , with a wild-type survival of 1% or higher, binding to DNA is 100-fold and to RNA 10- to 20-fold more efficient than that to protein. Between 20% and 65% of the 8-MOP binds to macromolecules that are neither nucleic acids nor protein. The number of DNA-bound 8-MOP molecules for the haploid genome rises from 14 (unirradiated control) to 349 at the highest UVA exposure dose (60 kJ M −2 ). Gel chromatography reveals three types of DNA thymine photoproduct, the pyrone-side monoadducts, the furan-side monoadducts and the diadducts. Among these, pyrone-side monoadducts always constitute the smallest fraction, regardless of whether the treatment is with in vitro or in vivo 8-MOP plus UVA.

The genes SNQ and SFA confer hyperresistance to 4-NQO and FA when present on a multi-copy plasmid in yeast. Both are non-essential genes since transplacement of SNQ by a disrupted snq-0::LEU2 yielded stable and viable haploid integrants. Southern analysis revealed that SNQ and SFA are single-loci genes, and OFAGE analysis showed that they are located on chromosome XIII and IV, respectively. Northern blot analysis of SNQ and SFA revealed poly(A)+ RNA transcripts of 2 kb and 1.7 kb, respectively. Nuclease S1 mapping showed SNQ to have a coding region of 1.6 kb and SFA, one of 1.3 kb. The 5' coding regions were determined for both genes, while the 3' end could only be determined for gene SNQ. Both genes do not appear to contain introns. The SFA locus was also mapped by transposon mutagenesis. Tn10-LUK integrants disrupted the SFA gene function at sites that were determined by subcloning to lie within the SFA transcription unit.

The formation and removal of formaldehyde-mediated DNA protein cross-linking was measured by CsCI density gradient analysis in yeast strains of differing resistance to formaldehyde. Wild-type cells and transformants made hyperresistant to formaldehyde by a multi-copy vector containing the yeast SFA gene were specifically labeled in their DNA and incubated in the presence of formaldehyde. Treatment with formaldehyde lead to the formation of equal amounts of DNA protein cross-links; subsequent liquid holding of cells for 24 h resulted in the removal of nearly all DNA protein crosslinks regardless of the original formaldehyde resistance status of the strains.

The three haploid yeast strains T2tmpl1-3, T2tmp1-1, and T6tmp1-51 auxotrophic for 5'-dTMP differ in their requirement for thymidylate: 72, 16, and 3 mug 5'-dTMP/ml will restore optimal growth, respectively. Thymidylate low requirement in strain T2tmp1-1 and T6tmp1-51 is termed tlrA and tlrC, respectively. When the growth medium is made 5 x 10(-4) M for 5'-dTMP only strain T6tmp1-51 is severely inhibited in RNA and DNA synthesis. This inhibition is reversible after removal of excessive 5'-dTMP. The inhibitory characteristic is in marked contrast to "thymineless death" due to the lack of 5'-dTMP in strain T6tmp1-51 where only DNA synthesis stops while RNA synthesis continues. The inhibitory effect of 5 x 10(-4) M 5'-dTMP is not due to the 5'-dTMP auxotrophy but to the thymidylate low requiring character (tlrC) in strain T6tmp1-51. The arrest of RNA and DNA synthesis by high concentrations of exogenous 5'-dTMP suggests a regulatory role of either the mono- or triphosphate on nucleoside or nucleotide biosynthesis in yeast.

Treatment of sensitive cells of Saccharomyces cerevisiae with killer toxin KT28 affected cell viability after 2 h: the effect was dependent upon the availability of a utilizable energy source. Treatment led to an interruption of cell growth. The mother cells contained nuclear DNA, whereas their daughter buds did not. Using a killer-toxin-sensitive thymidine auxotroph of S. cerevisiae carrying a temperature-sensitive thymidylate uptake mutation, it was shown that the incorporation of dTMP at the permissive temperature was inhibited within 30 min of the addition of KT28. When cells labelled at the permissive temperature were incubated at the restrictive temperature, the level of radioactivity declined in the absence but not in the presence of KT28. No other effects of KT28 were observed within 2 h of its addition, and it is concluded that the inhibition of DNA synthesis is an early effect of the action of KT28.

The method for assaying thymine-containing dimers in yeast is based on highly efficient ([3H]-deoxythymidine-5′-monophosphate) DNA-specific labelling and employs ascending thin layer chromatography. It allows satisfactory quantitative analysis down to UV-doses of 500 erg/mm2.

When dTMP in concentrations greater than 100 microM is offered to growing cells of thymidylate low-requiring yeast strains it is both mutagenic and toxic. At exposure concentrations greater than 1 mM dTMP interferes significantly with the low-affinity phosphate permease even in the presence of exogenous phosphate concentrations of 6 mM. Chemical analysis and 31P NMR spectroscopy reveal that excess dTMP disturbs phosphate metabolism in thymidylate low-requiring strains but not in the wild type. The most prominent changes in phosphorus-containing molecules are found in polyphosphates of which up to 20% are broken down within a 20-min time span with a concomitant increase in orthophosphate pools.

dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae. Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis. Still higher dTMP concentrations are primarily cytotoxic. The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light. Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity. Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.

Mustard gas induces inactivation and mutation in yeast. Both effects are dose-proportional, indicating single-hit events. Induction of both effects is influenced by the cell's capacity for DNA dark-repair, whereby the probability of reversion is highest in repair-proficient cells. Binding of mustard gas to cells and probably to DNA is independent of DNA-repair systems. The number of inter-strand cross-links, as determined by assaying for renaturability of alkalidenatured DNA, increases in a dose-proportional manner. At 37% survival an excision-deficient strain contains 55 inter-strand cross-links. Chromatographic analysis yields several alkylation products of DNA. Their relative frequencies resemble the values reported for E. coli and bacteriophage T7.

Strain 211-1aM of Saccharomyces cerevisiae was found to specifically incorporate into its nuclear and mitochondrial DNA exogenous 5′-dTMP when it is incubated in a synthetic medium N rich in inorganic phosphate. 3′-digestions of DNA from cells labelled with 32P-5′-dTMP revealed that the 5′-dTMP molecules pass through cell walls and membranes to the sites of DNA synthesis without being broken down. It is possible in medium N to generate DNA which almost totally derives its thymine-contents from external sources when rather high concentrations of 5′-dTMP (approx. 100 μg/ml) are offered.

Selection of mutants of Saccharomyces cerevisiae sensitive to the DNA cross-linking agent nitrogen mustard (HN2) at two temperatures (23 °C and 36 °C) yielded two isolates with thermoconditionally enhanced (ts) sensitivity to the mutagen. Both were due to single recessive nuclear genes. Mutant allele snm1–2 ts showed mainly ts-sensitivity to HN2, whereas mutant allele snm2-1 ts conferred ts-sensitivity to HN2, half mustard (HN1) and UV. In temperature-shift experiments it was determined that the functions of SNM1 and SNM2 are needed for recovery within 6 to 7 h. after mutagen exposure during incubation at 23 °C on YEPD when HN2 and UV are applied. After HN1 treatment the SNM2 coded function is required for recovery for about 14 hrs. This possibly indicates a handling of UV- and HN2-induced lesions different from that of HN1-induced lesions.

Cells of Saccharomyces cerevisiae strain 211-1atmp1-1 which are auxotrophic for 5′-dTMP exhibit “thymineless death” (TLD) when deprived of the nucleotide. After an initial lag of about one generation time cells lose viability in exponential fashion halving their titer every 90 min. Thymine and thymidine (100 μg/ml) cannot prevent TLD in the absence of 5′-dTMP. Although the cell titer is constant during 24 hrs of 5′-dTMP deprivation, the cell mass synthesized increases by a factor of six during this period. Budding is stopped and cells attain a swollen shape. Synthesis of RNA and protein does occur in cells deprived of 5'-dTMP, the rates of synthesis being significantly lower than those in the controls.

Reversion of two nuclear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (rad1 rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (radl rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom.

Although strain 211-1 aM is able to take up 5′-dTMP it cannot overcome growth-inhibition caused by an aminopterin (APT)-sulfanilamide (SAA)-mediated block of thymidylate biosynthesis in the presence of exogenous 5′-dTMP. 11 mutants (T-strains) of strain 211-1 aM were isolated being able to grow under these conditions. Genetical analysis of the T-strains revealed in each case the mutation of a single recessive gene, TYP, to be responsible for this character, and that the mutated genes belong to three complementation groups. One such typ-mutant, strain 211-1 aMT2, studied in detail was found to utilize exogenous 5′-dTMP as poorly as strain 211-1 aM. However, when thymidylate-biosythesis is inhibited by APT plus SAA the utilization of 5′-dTMP is increased by a factor 2.5 as compared to conditions where the antimetabolites are absent. Next we isolated a typ-mutant- derivative (typ tlr- mutant) utilizing exogenous 5′-dTMP better by a factor 2.5 than strain 211-1 aMT2. This typ tlr- mutant was made auxotrophic for 5′-dTMP. The efficiency of utilization of exogenous 5′-dTMP found for these typ tlr tmp-mutants was the same as that of their typ tlr- parental strain in the presence of APT plus SAA.

A screening procedure is presented which allows the isolation of yeast mutants (typ tlr) with highly efficient utilization of exogenous deoxythymidine-5′-monophosphate (5′-dTMP) (>50% ). Data are given concerning the phenomenon of 5′-dTMP utilization in general: (i) The ability of S. cerevisiae to incorporate exogenous 5′-dTMP was found to already be a wild type feature of this yeast, i. e. apparently not to be due to any mutation such as typ , tup, tmp per or tum. Consequently these mutations are interpreted as amplifiers of a pre-given wild type potency. So far eight stages of 5′-dTMP utilization were detected as classified by the optimal 5′-dTMP requirement, with 5′-dTMP biosynthesis blocked, of the corresponding mutant strains isolated. All of them fit well into a mathematical series of the type “2n × 1.5” (n = 0, 1, 2, … , 11), where the product term for n = 11 represents the 5′-dTMP requirement (μg/ml) of the best 5′-dTMP utilizing wild type strain found, (ii) Amplification of the 5′-dTMP utilizing potency obviously is due to any genetically determined alteration of the yeast 5′-dTMP uptaking principle itself or of physiological processes accompanying the monophosphate’s uptake, (iii) The functioning of 5′-dTMP uptake requires acidic (≦ pH 6) conditions in the yeast cell’s outer environment, (iv) Some yeast typ and typ tlr mutants were found to exhibit a more or less pronounced sensitivity towards exogenously offered 5′dTM P. The response of a sensitive strain towards inhibitory concentrations of the nucleotide apparently is co-conditioned by the presence or absence of thymidylate biosynthesis. With 5′-dTMP biosynthesis blocked the 5′-dTMP mediated inhibition is a permanent one and finally leads to the death of a cell. With a functioning thymidylate biosynthesis, in contrast, the inhibition is only temporary, (v) Yeast typ or typ tlr strains were observed to dephosphorylate exogenous 5′-dTMP to thymidine due to a phosphatase activity which cannot be eliminated at pH 7 + 70 mм inorganic phosphate conditions in the growth medium. This 5′-dTMP cleavage obviously occurs outside the cell and does not seem to be correlated both to the monophosphate’s uptake and to the phenomenon of 5′-dTMP sensitivity. The destruction of 5′-dTMP does not disturb (5′-dTMP) DNA-specific labelling.

A method to detect low levels of interstrand cross-links in DNA of Saccharomyces cerevisiae is described. Isopycnic ultracentrifugation of alkali-treated, unpurified Eaton press homogenates allows the detection of less than one cross-link per yeast chromosome. Efficient separation of single-and double-stranded DNA requires low cell density and addition of glycerol during homogenization. Using a yeast strain defective in excision repair, a dose dependent formation of interstrand cross-links after treatment of cells with biological doses of nitrogen mustard. Triaziquone and Chloramubil could be demonstrated. The most powerful of these alkylating agents is Triaziquone: half of the DNA molecules are shown to be cross-linked after a 12 min exposure to 9×10-9 g/ml of the drug. The cross-linking reaction continues after excessive alkylating agent is removed. After having reached a maximum the fraction continues after excessive alkylating agent is removed. After having reached a maximum the fraction of renaturable DNA decreases upon further incubation. The speed of this “after-reaction” depends on temperature: 48 h after the end of treatment renaturability of DNA has almost completely disappeared when cells are kept at 36° C.

It is shown that highly efficient utilisers of exogenous dTMP of the yeast Saccharomyces cerevisiae are able to excrete the nucleotide with similar efficiency. Strains Pi-repressible in acid phosphatase/nucleotidase excrete dTMP at extracellular high Pi; strains constitutive for this enzymic activity excrete dThd. Excretion of thymidylate and dThd, unlike uptake of exogenous dTMP, seems to be unaffected by the extracellular pH, by the extracellular presence of dTMP, and to be rather independent of the extracellular presence of a metabolisable carbohydrate such as D(+)-glucose. A model of the yeast dTMP-incorporation principle (TIP) is presented suggesting that it is also responsible for export of endogenous thymidylate.

The cytotoxic and DNA-damaging effects of cyclophosphamide (CP) and its ‘activated’ derivative 4-OOH-CP were studied using a series of strains of S. cerevisiae which allow a phenotypical classification of genotoxic characteristics as well as direct physicochemical demonstration of key DNA lesions. The concurring results of biological and biochemical experiments indicate that (i) non-activated CP has a weak but detectable monofunctional alkylating potency, leading to DNA strand breaks and (ii) 4-OOH-CP has the ability to induce both DNA strand breaks and interstrand cross-links. The activity of CP is probably due to spontaneous decomposition in aqueous solution.

The time course of REV2 dependent recovery from prelethal UV damage and UV-induced locus-specific reversion of the his5-2 allele was determined in temperature-shift experiments by use of a thermoconditional allele of the rev2 gene (rad5-8, rev2ts). In UV-irradiated, exponentially growing rev2ts cells the REV2 dependent repair activity persists for up to 8 h at permissive temperature (23 degrees C), while the REV2 dependent mutagenic process is mostly completed within 2 h. The REV2 dependent process in exponentially growing cells is highly impaired by inhibition of protein synthesis. However, a REV2 dependent repair activity independent of de novo synthesis is detectable, even in the presence of up to 200 micrograms/ml cycloheximide, a response not found in stationary phase cells. Thus, the REV2 dependent process seems to be partially constitutive in exponentially growing cells. Additionally, exponentially growing rev2ts cells were considerably more UV-sensitive at restrictive temperature (36 degrees C) than were stationary phase cells.

Extending previous work with E. coli and mammalian cells in culture, forward-mutation frequencies induced by ethyl methanesulfonate (EMS) were quantitatively compared in Neurospora crassa and Saccharomyces cerevisiae under standardized conditions. Concomitantly, the actual dose to DNA was measured by determining the amount of radioactivity bound to DNA after treatment with tritium-labeled EMS. After exposure to EMS (2.5–50 mM), alkylation levels in N. crassa and S. cerevisiae were similar to those previously determined in E. coli and cultured mammalian cells. Consistently, there was a slightly less than proportional increase of the DNA alkylation level with the exposure concentration of the mutagen. Forward mutagenesis induced in yeast and N. crassa showed exponential kinetics with exponents of 1.5 and 2.6, respectively. These results are similar to those previously reported with E. coli, which differed from the results with cultured mammalian cells, where a linear dose-effect relationship between exposure and genetic effect was observed. These differences may reflect differences in the fate of EMS-induced adducts by cellular DNA repair systems, but are not due to initial differences in DNA alkylation levels. The fate and persistence of specific DNA adducts potentially responsible for pre-mutagenic changes are under investigation.

n/a

Extracellular treatment with methylene blue and light (MB+Li) caused inactivation and induction of mutations in Serratiaphage ϰ. Inactivation is partly due to lesions in the DNA as concluded from marker rescue (CR) experiments where some 67% of UV-and 40% of MB+Li-induced damage could be reactivated. About 32% of the lethal lesions after MB+Li could be reactivated by host cell reactivation (HCR) whereas induced mutations were not influenced. Photoreactivation (PR) of lethal damage and mutations was not detected. Postincubation of MB+Li-inactivated free phage in saline or dye solution at 30°C caused additional inactivation but mutations again were not influenced. The 3 types of plaquetype-mutations scored (clear=c+1, narrow=e, and pale=b) were induced by MB+Li according to concave light-dose curves indicating induction by different average hit numbers (2.3, 1.6, and 3.0). Increase of the fraction of plaquetype-mutants due to selection was excluded. Increase of the mutant types “g”, however, was due to selection only. The proportion of the 3 mutant-types induced by MB+Li was different (at all light doses) from the ones induced by the guanine-specific mutagens EMS or low pH; thus the MB+Li-induced lesions leading to mutation are due to different changes of guanine moieties or do not have their origin in guanine destruction at all.

n/a

Strain 211-1a of the yeast Saccharomyces sp. allows the specific labelling of nuclear and mitochondrial DNA by exogenously supplied radioactive thymidine monophosphate (TMP) during the latter half of log phase growth. Genetic analysis of crosses between derivatives of strain 211-1a and wild-type strains that do not take up TMP showed a 2:2 segregation pattern indicating that a single gene controls the uptake of TMP. The allele allowing the uptake of TMP is recessive in that diploid strains take up TMP only in the homozygous configuration.

n/a

n/a

In the simple eucaryote Saccharomyces cerevisiae there are at least three phenotypically distinct classes of mutants sensitive to inactivation by radiations and alkylating agents: class I mutants are sensitive to ultraviolet light and nitrogen mustard (HN2); class II mutants are sensitive to X-rays and methylmethane sulphonate (MMS); and class III mutants are sensitive to all four of these agents. We have constructed doubly mutant strains of types (I, I), (I, II), (I, III), and (II, III) and have measured their sensitivity to UV, X-rays, HN2 and MMS in order to characterize the interactions of the various mutant gene pairs. Class (I, III) double mutants proved to be supersensitive to UV and HN2 and class (II, III) double mutants proved to be supersensitive to X-rays and MMS. All other double mutants showed little or no enhancement of sensitivity over their most sensitive single mutant parents. Mutants of class I are known to be defective in excision repair and our results are consistent with the idea that there exist at least two additional pathways for dark repair in yeast, one capable of repairing X-ray and MMS damage to DNA, and another, possibly analogous to post-replication repair in bacteria, that competes with the other two for damaged regions in DNA.

Double mutants containing the radiosensitive alleles uxsl (UVR-EXR-) and either uvsl-2 or uvsl 9-2 (both UVR-EXR+) have been constructed. We find that these double mutants are extremely sensitive to UV: their LD37 doses are 0.3–0.5 ergs/mm2 as compared to 16–40 ergs/mm2 for the radiosensitive parental strains carrying only a single mutant gene and 456 ergs/mm2 for wild-type.

The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids. Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements. The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate. No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate. Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type. Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair.

Activated cyclophosphamide (CP) induces DNA interstrand cross-links and DNA strand-breakage in the chromosomes of yeast. In contrast to the post-incubation reaction of other polyfunctional alkylating agents, a further increase of cross-links after treatment with activated CP was hardly detectable. This is possibly due to the observed continued DNA fragmentation which decreases the sensitivity of the cross-link assay. Cell killing by non-activated CP depends on the function of DNA repair as well as on the mode of cellular energy metabolism. At concentrations 1000-fold higher than those used for activated CP it induces DNA single- as well as double-strand breaks. In contrast, no induction of interstrand cross-links could be detected.

Using the thermoconditional yeast mutant rev2ts that controls an apparently site-specific step of mutagenic DNA repair it was possible to measure the time course of REV2 dependent UV-induced reversion of the ochre allele his5-2 and recovery of survival for UV-treated stationary phase cells: due to the rev2ts coded protein being active at 23 degrees C, survival and mutation frequencies increased with duration of incubation under permissive conditions in growth medium before the temperature was shifted to 36 degrees C (restrictive temperature). This increase was abolished in the presence of the protein synthesis inhibitor, cycloheximide. Furthermore, the REV2 dependent recovery of survival could be blocked or nearly blocked by cycloheximide added at any time during repair. Therefore, REV2 dependent repair can be characterized as a process requiring concomitant protein synthesis. These findings give further support to the concept that in yeast, mutagenesis involves UV inducible components of DNA repair.

A discrete class of strains of Saccharomyces cerevisiae, able to utilize, highly efficiently, exogenous deoxythymidine-5'-monophosphate (dTMP),was found to be sensitive to concentrations greater than 10 Micro M dTMP in an otherwise complete growth medium. Excess dTMP is cytostatic and cytotoxic: 90% of exponentially growing cells lose colony forming ability within 1 h of exposure to excess dTMP is a growth medium. Uptake of dTMP, adenine, histidine, and leucine does occur during this thymidylate excess death (TED). dTMP is anabolized to higher phosphorylated synthesis is blocked under TED-conditions but not RNA and protein biosynthesis.

The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals. Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active. The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.

Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 (ts) were found epistatic to genes of the rad3 group, whereas snm2-1 (ts) was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.

Acid hydrolysis of purified DNA extracted from cells of a haploid repair-proficient ( RAD ) yeast strain that had been treated with 8-MOP + UVA revealed the existence of two major and one minor thymine photoproduct. At survival levels of the RAD strain between 100% and 1% furanside monoadducts constituted the major DNA lesion, followed by diadducts that, at the lowest survival level, nearly reached 50% of the thymine photoproducts; pyrone-side monoadducts were only detectable at the highest UVA exposure dose applied and clearly constitute a minority photoproduct. The number of induced diadducts was verified by determination of interstrand cross-links via denaturation and renaturation of 8-MOP + UVA-treated DNA from RAD and rad2 yeast strain. 8-MOP + UVA was shown to induce two types of locus-specific mutations: reversion of the lys1-1 ochre allele was between 20- to 50-fold higher than that of the his4-38 frameshift allele. Mutant yield for the lys1-1 reversion was the same in RAD and excision repair-deficient rad2-20 strains whereas frameshift mutagenesis was about eightfold higher in the rad2-20 background.

In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

An improved method for isolation of yeast m utants auxotrophic for 5′-dTM P is presented. The procedure employs the two folic acid antagonists am inopterin and sulfanilam ide (SAA). Selectiveness of the procedure depends on concentration of SAA and time of incubation. 44 mutants auxotrophic and 3 conditionally auxotrophic for 5′-dTMP were isolated. All belong to one complementation group. The corresponding gene was designated TMP1. Tetrad dissection revealed its chromosomal nature. TMP1 is not closely linked to the genes ADE2,, LEU1, ARG 4, ILV2, HIS5, LYS1 and the mating type locus. With the centromere-linked genes ARG4 and LEU1 I gene TMP1 exhibited second division segregation frequencies of 0.42 and 0.53 respectively, indicative of centromere-linkage. Strains auxotrophic and conditionally auxotrophic for 5′-dTM P were all respiratory deficient (petite). Genetical analysis indicates that the petite phenotype is due to loss of the rho factor in cells harbouring either tmp1 or tmp1ts alleles.

Of two mutant genes (snm1-2 (ts) and snm2-1 (ts)) conferring thermoconditional mutagen sensitivity in Saccharomyces cerevisiae one (snm2-1 (ts)) is shown to be centromere-linked. At the restrictive temperature this allele reduces UV-induced back mutation frequency of the ochre allele hiss-2 but has no influence on forward mutation at the CAN1 locus. Complementation tests and recombination analysis revealed snm2 (ts) to be allelic with rad5 (rev2).

Cyclophosphamide (CP), whether applied in its chemically activated form as 4-hydroperoxy-cyclophosphamide (4-OOH-CP) in vitro or in the host-mediated assay (HMA) using rats, exhibits toxic and mutagenic effects on excision deficient yeast cells. The expression of these effects is examined during a prolonged postincubation in buffer and compared with the ability of activated CP to induce interstrand cross-links and DNA fragmentation. At comparable doses, we observe a close similarity of biological and biochemical effects in either test system.

Treatment of yeast cells with 4-hydroperoxy-cyclophosphamide (4-OOH-CP), the chemically activated form of cyclophosphamide, results in cell killing, induction of DNA interstrand cross-links and DNA fragmentation. Toxicity of 4-OOH-CP is greatly influenced by the cell's capacity of DNA dark-repair: genetic blocking of non-epistatic pathways of DNA repair results in an increase of sensitivity of several orders of magnitude. DNa primary lesions have been measured using a haploid, excision deficient, dTMP-uptaking mutant of S. cerevisiae. In this strain, a significant extent of DNA cross-linking can already be observed at a survival of 88%. At a concentration of 100 nmol/ml 4-OOH-CP, renaturability of DNA increases up to 12 h of drug exposure and drops to lower values upon further incubation. In contrast to the time course of renaturability, DNA double-strand breakage is seen at later stages of drug treatment and continuously increases as a function of incubation time. Whereas inactivation of cells and induction of strand breakage continue upon postincubation of cells, comparable effects are much less pronounced for DNA renaturability.

Four haploid yeast strains differing in proficiency for DNA repair were treated with cis- or transDDP. The wild type was least sensitive while the excision-deficient mutants rad1, rad2 and snm1 exhibited higher sensitivities to either platinum compound. In all four strains tested cisDDP showed a two- to five-fold higher cytotoxicity than equimolar concentrations of transDDP. DNA interstrand cross-linking was caused by both agents in all strains. However, transDDP introduced more DNA cross-links at exposure times up to 6 h while cisDDP was the more active cross-linking agent at longer times. There was no clear-cut correlation of the number of DNA interstrand cross-links with survival. Formaldehyde-treated cells showed DNA with lower buoyant density due to proteinase K sensitive DNA-protein cross-linking; this effect was not observed after treatment with either platinum compound. Post-treatment incubation of wild-type cells exposed to cisDDP led to degradation of DNA by single and double-strand breaks, parallel with further increase of DNA interstrand cross-linking. DNA from transDDP-treated cells did not show extensive degradation although interstrand cross-links were lost during liquid holding.

The increasing use of several different organisms (1, 2) to detect, verify, quantify, and extrapolate the potential mutagenic and carcinogenic effects of environmental chemicals (3) makes a critical evaluation of their sensitivity and reliability necessary. Of special interest is the determination to what extent the response to chemical mutagens is influenced by intrinsic cellular factors, such as chromosome structure and DNA content per cell, and also by metabolic and physiologic differences between test organisms or within strains of the same species (4). A quantitative comparison of the induced genetic effects first requires a precise determination of the actual dose at the target molecule, say, DNA. The reason for individual dose measurements in the different indicator organisms is that one cannot, a priori, assume that identical exposure concentrations of a chemical applied to the system will lead to identical dose at the DNA level. Instead, one expects that penetration and metabolism in general will influence the extent of reaction of the compound with DNA.

In haploid and diploid S. cerevisiae the dimer yield ratio TT̂/CT̂ is found to be 1.2/1 and 1.3/1, resp., at the UV (254 nm) unit dose 1 erg/mm2, the share of TT̂ and CT̂ in a UV (254 nm) lethal hit being 0.7 TT̂ and 0.6 CT̂. A general formulation of the UV lethal hit is given and discussed. The TT̂ + CT̂ yields obtained for S. cerevisiae are compared to those reported for other organisms. It is found that there obviously exists a directly proportional linear correlation between genome size and TT̂ + CT̂ yield for the UV dose range well below the stationary levels of the TT̂ and CT̂ formation kinetics.

Two different UVA irradiation systems were initially biologically calibrated with two haploid yeast strains proficient and deficient, respectively, in nucleotide excision repair. The number of DNA lesions introduced into the cell's genome by the photoactivated bifunctional furocoumarin 8-MOP was then calculated by means of the applied UVA exposure doses. At LD37 the repair-proficient wild type had about 14 ICL and 34 furan-side monoadducts in its DNA, while doubly blocked repair mutant rad3-12 pso1-1 had 2 ICL and 3 monoadducts. Locus-specific reversion of lys1-1 followed two-hit kinetics in the repair-proficient wild type and one-hit kinetics in an excision-deficient rad2-20 mutant, as would be expected if ICL was the main type of mutagenic lesion in the wild type and monoadducts the main mutagenic lesion type in the excision-deficient strain. Quantitative comparison of 8-MOP + UVA-induced ICL with those induced by bifunctional mustard revealed the former to have a much higher genotoxicity.

Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties. The screening technique, based on HN2-sensitivity as sole criterion, yields approximately 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain. Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups. They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light.