Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells.

DNA replication in ultraviolet-irradiated human cells was examined by treatment of the extracted DNA with a single-strand specific endonuclease from Neurospora crassa. WI38 cells were uniformly labeled with 32Pi for two generations before irradiation and then labeled with [3H]thymidine after irradiation. The isolated DNA was sedimented in neutral sucrose gradients after incubation with the endonuclease. The endonuclease treatment had no effect on the sedimentation profiles of either [32P]DNA or [3H]DNA from unirradiated control cultures. The endonuclease treatment also did not significantly alter the profile of [32P]DNA from irradiated cultures but did introduce breaks in the 3H pulse-labeled DNA synthesized after irradiation. These results indicate that DNA synthesis after ultraviolet irradiation proceeds in such fashion that gaps are formed along the newly made strand, leaving regions of single strandness in template DNA. As replication proceeds these gaps disappear and 2 h after irradiation (100-250 ergs/mm2) they are barely detectable by the endonuclease assay.     

A chicken repetitive DNA sequence that is highly sensitive to single-strand specific endonucleases

A DNA sequence consisting of the 5-mer AGAGG repeated tandemly 32 times has been detected in a chicken genomic clone and found to be present in about 2000 copies per chicken genome. This sequence was highly susceptible to single-strand specific endonucleases isolated from Aspergillus oryzae (S1) and mung bean, but cleavage by a single-strand specific endonuclease isolated from Neurospora crassa occurred only at a pH below 5.5. Endonucleolytic cutting of the AGAGG sequence by the single-strand specific enzymes required a supercoiled substrate and was independent of ionic strength.     

Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suc ⁰ and N. crassa inv strains transformed with p NC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suc ⁰ ( p NC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa , although S. cerevisiae suc ⁺ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI -restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv^- to Inv⁺ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases

The functional significance of mono-, di-, and tri-methylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys(9) (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe(281) of Neurospora crassa DIM-5 or Phe(1205) of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono- or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (-/-) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono- and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.     

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.     

Tests for the genotoxicity of m-AMSA, etoposide, teniposide and ellipticine in Neurospora crassa

The antitumor agents m-AMSA, etoposide, teniposide and ellipticine have been reported to be potent clastogens in mammalian cells but non- or weakly mutagenic in bacteria; these observations have been correlated to the interference of these chemicals with DNA topoisomerase II activity in the former, but not in the latter, organisms. The genotoxicity of these 4 agents was evaluated using ad-3 reverse- and forward-mutation tests in Neurospora crassa. These agents (up to 0.8 mumole/plate) did not cause reversion in conidia of the ad-3A frameshift strains N24 and 12-9-26 using the overlay plate test, as contrasted to the positive control frameshift mutagen ICR-170. Heterokaryon 12 (H-12) of N. crassa permits the recovery of all classes of forward mutation at the ad-3+ region, including multilocus deletions. Using resting conidia of H-12 in a suspension assay, ellipticine was moderately mutagenic but no increase in ad-3 mutants was noted with the other 3 agents at a dose of 100 micrograms/ml. In vegetative cultures of H-12 grown in the presence of these agents, all 4 agents were nonmutagenic at a dose of 100 micrograms/ml. The positive control mutagen ICR-170 was mutagenic in both resting conidia and growing cultures of H-12. A similarity between the topoisomerase II of N. crassa and DNA gyrase of bacteria is suggested.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Ribosomal RNA genes of Neurospora crassa: multiple copies and specificities

Ribosomal RNA genes were isolated from the germinated conidial and mycelial cells of N. crassa by repeated cycles of 3H-DNA:rRNA reactions followed by hydroxyapatite chromatography. Specificity of multiple copies of those rDNAs with respect to N. crassa cell types was studied. The fraction of N. crassa germinated conidial in vitro labelled 3H-DNA recovered in the presence of rRNA isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rDNAs obtained in mycelial cells. These isolated rDNAs reacted specifically to 26S and 17S rRNAs of eukaryotic (N. crassa) organisms and did not react with 4S tRNAs. rRNA:rDNA reassociation kinetics studies indicate that 90% of the rRNA genes were homogeneous and not identical with the other 10% rRNA genes isolated from N. crassa mycelia. These studies suggest that the possible heterogeneity of rDNA sequences of N. crassa cannot be attributed to inclusion of any tDNA sequences as has been shown in the heterogeneity of rDNA sequences of the bacterium Escherichia coli. The heterogeneity of multiple copies of N. crassa rDNAs could be due to differences in internal or external spacer regions of N. crassa rRNA genes.     

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.     

Enrichment for auxotrophic and heat-sensitive mutants of Neurospora crassa by tritium suicide.

Tritium suicide is shown to be an effective technique for mutant enrichment in Neurospora crassa. When mutagenized conidia were labelled to a high specific radioactivity either with a tritiated amino acid mixture or with [5-3H]uridine at a non-permissive temperature and stored at 4 degrees C to accumulate decays, there was a 13-15 fold enrichment for temperature-sensitive mutants relative to the original mutagenized cultures. For a wild type culture of Neurospora crassa labelled with [5-3H]uridine at 35 degrees C the probability of cell killing per tritium decay was calculated to be 3.64 X 10(-5).     

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.     

Nucleotide excision repair and photoreactivation in the entomopathogenic fungi Beauveria bassiana, Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus and Verticillium lecanii

AIMS: To compare the DNA repair capabilities of the entomopathogenic fungus (EPF) bassiana to the EPF Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii, and the fungi Aspergillus niger and Neurospora crassa. METHODS AND RESULTS: Germination of B. bassiana conidiospores following ultraviolet (UV) irradiation was used to show that nucleotide excision repair and photoreactivation decrease the post-UV germination delay. These two modes of repair were characterized and compared between the aforementioned EPF, A. niger and N. crassa using a physiological assay where per cent survival post-UV irradiation was scored as colony forming units. CONCLUSIONS: The results showed B. bassiana and M. anisopliae are the most UV-tolerant EPF. The DNA repair capabilities indicated that EPF do not have all DNA repair options available to fungi, such as A. niger and N. crassa. SIGNIFICANCE AND IMPACT OF THE STUDY: A key factor detrimental to the survival of EPF in agro-ecosystems is UV light from solar radiation. The EPF literature pertaining to UV irradiation is varied with respect to methodology, UV source, and dose, which prevented comparisons. Here we have characterized the fungi by a standard method and established the repair capabilities of EPF under optimal conditions.     

Enzymatic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.     

Generation of Benomyl Resistant Beauveria bassiana Strains and Their Infectivity Against Helicoverpa armigera

Beauveria bassiana transformants were obtained by conventional protoplasting and transformed by eletroporation and polyethylene glycol (PEG) treatment. These displayed mitotic stability in Beauveria bassiana . Strains transformed with pSV50 harbouring the β-tubulin gene of Neurospora crassa grew well on benomyl concentrations of 10 μg ml -1 unlike the recipient strain. The transformants were mitotically stable on either selective or non-selective medium. The efficiency of transformation by linear and circular pSV50 cosmid was 8 and 10 transformants per mug DNA per ml viable protoplast by electroporation, respectively, and 4 and 6 by the protoplast PEG method, respectively. Southern blot and hybridization of undigested fungal DNA of wild type and four transformants, probed with β-tubulin sequence of pSV50, showed hybridization at high M r region of genomic DNA in four transformants, whereas in wild type genomic DNA, no homology of the sequence was observed. Digested genomic DNA, of four transformants gave a complex hybridization pattern. Virulence tests of the transformants showed that there was no significant loss in the pathogenicity toward Helicoverpa armigera third instar larvae. This method of transformation should prove useful with entomopathogenic fungal species in which a genetic transformation system has not yet been established.