Deoxyribonucleoside triphosphate pools in vitamin B-12-deficient Euglenagracilis.

The size of the deoxyribonucleoside triphosphate pools of vitamin B-12-deficient cells of Euglena gracilis, and of vitamin B-12-deficient cells repleted with the vitamin, were measured. We found that the pools were very small, if they exist at all, in deficient cells but expand rapidly with the addition of the vitamin. The sizes of the pools decrease when DNA synthesis is completed, and are very small when the cells begin to divide.     

Urease defective mutants in Neurospora crassa

Summary A method for isolating urease mutants was developed. It is based on the use of microconidial strains with small and compact colonies. Mutants are detected by their inability to change the color of a pH indicator when they are brought in contact with a solution of urea. The assay is performed in the absence of growth conditions so that the colonies remain separate. Two isolated urease mutants are unable to grow on urea as the sole source of nitrogen, but grow as well as the wild type on other sources of nitrogen. The same two mutants give rise to different acidities in liquid growth medium. The two mutants are also genetically different (K?lmark, 1969 b). The finding that two genetically and physiologically distinct loci participate in the control of one enzyme is discussed.     

Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.     

Epistasis, photoreactivation and mutagen sensitivity of DNA repair mutants upr-1 and mus-26 in Neurospora crassa

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.     

Isolation of putrescine-requiring mutants of Neurospora crassa

Two auxotrophs of Neurospora crassa have been isolated that give a positive growth response to putrescine, spermidine or spermine. One of the mutants is deficient in ornithine decarboxylase activity and has been designated put-1. Both mutants map on linkage group VR, fail to complement and are infertile when crossed to one another, indicating that they are probably alleles. A putrescine auxotroph is incapable of suppressing a pro-4 mutant. The isolation of the mutants confirms that putrescine is an essential factor for the normal growth of the organism, and is synthesized via a single pathway in Neurospora.     

Complementation among cytoplasmic mutants of Neurospora crassa

Summary Heteroplasmons with normal growth rates are formed when the slow-growing, female fertile, group I or II extranuclear mutants of Neurospora crassa are combined by forced heterokaryosis with the female sterile, stopper mutants of group III. Different mutants from the same growth and fertility group do not complement each other, and the poky-like strains of group I do not interact synergistically with [mi-3], the only known group II mutant. The mitochondrial cytochrome system of the complementing heteroplasmons are as abnormal as the cytochrome complements of the component extranuclear mutants, indicating that defects in the electron transport system represented by those mutants are related inconsequentially to growth. The observed functional complementation indicates the expression of the mitochondrial genome is not restricted to the specific organelle of which it is a part.Contribution No. 1255 Department of Agronomy; Contribution No. 1148, Division of Biology, Kansas Agriculture Experiment Station, Manhattan, Kansas.     

Deoxyribonucleoside triphosphate pools in regenerating rat liver: effect of hydroxyurea and exogenous deoxypyrimidines

Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals.     

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria.

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.     

Neurospora crassa supersuppressor mutants are amber codon-specific

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.     

A new class of p-fluorophenylalanine-resistant mutants in Neurospora crassa

A p-fluorophenylalanine-resistant mutant (acc ^phe ) which grows on minimal medium has an altered prephenate dehydrogenase and maps at the try-1 locus. Two other tyr-1 mutants which require tyrosine for normal growth can eventually grow on minimal or minimal plus p-fluorophenylalanine (FPA). The three different tyr-1 mutants all accumulate phenylalanine when incubated in minimal medium. FPA is incorporated into protein at only 10–15% the wild-type rate when mutant conidia are incubated in a minimal salts-glucose system. Under the same conditions, phenylalanine incorporation in the mutants is initially the same as in wild type. When tyrosine is included in the medium, resistance to FPA is lost, phenylalanine accumulation is prevented, and FPA is incorporated into protein at the wild-type rate. Tyrosine apparently prevents the overproduction of phenylalanine by preventing the overproduction of chorismate and prephenate.This work was supported, in part, by an Atomic Energy Commission grant to the Institute of Molecular Biophysics, the Florida State University, and by the Genetics Training Grant, funded by the National Institute of Health. It contains, in part, data from the doctoral thesis of the senior author, who was supported by a Florida State University Nuclear Fellowship and by a Public Health Service Fellowship.     

Biochemical basis of radiation-sensitivity in mutants of Neurospora crassa

The available UV-sensitive mutants of Neurospora crassa were examined for their ability to excise and photoreactive cytosine-containing dimers invivo. All strains exhibited in vivo photoreactivation, including upr-1, which was originally thought to be deficient in photoreactivation. Two strains, uvs-2 and upr-1 were shown to be deficient in excision repair; uvs-3 was shown to contain a residual amount of excision capabilit. The remaining strains, uvs-1, uvs-5, and uvs-6, were normal in their ability to excise dimers. Based on these results, tentative analogies were drawn between the Neurospora mutants and the known classes of UV-sensitive mutants in E. coli. Accordingly, the N. crassa mutants were classified as uvs-1, -lon; uvs-2, -uvr; uvs-3, -uvr (rec?); uvs-5, -lon; uvs-6, -rec; and upr-1, -uvr. A comparison was made between the biochemical responses and the available published data on mutation induction in the Neurospora mutants. Althoughsome relationships were seen between repair defects and mutation induction, too little data were available for any definitive conclusions.