pH homeostasis of the circadian sporulation rhythm in clock mutants of Neurospora crassa

The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq⁺) and the mutants chr, frq¹, frq⁷, and frq⁸. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4-9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq¹. A loss of pH homeostasis of the period, however, was observed in the mutants frq⁷ and frq⁸, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H₂PO₄^- ion has been found, indicating that the uptake of H₂PO₄^- may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq⁷ and frq⁸ mutants may be related to less easily degradable FRQ^7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ⁷ protein degradation increases and should lead to shorter period lengths. (Chronobiology International, 17(6), 733-750, 2000)     

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.     

Kinetics of circadian band development in Neurospora crassa

The circadian rhythm of Neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. To follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. The circadian behavior of N. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultivation in conventional Petri dish cultures. Two different growth parameters were measured: total colony mass (true growth rate) and distance (colony radial expansion or hyphal elongation). Determinations of cellular mass revealed a dramatic circadian oscillation with a marked drop in growth rate during new interband formation followed by a sharp increase during the development of a new conidiation band. On the other hand, we found that the radial expansion of the colony previously reported to decrease periodically seemed unaffected by the circadian clock. Densitometric analysis showed no initial difference in the expanding margin of the colony, independent of whether that area was destined to be a band or an interband. The band areas increased rapidly in density for about 15 h whereas the interband areas maintained an equally rapid rate of increase for only 6h. The density of band areas kept increasing slowly for almost 40 h, along with an increase in the amount of conidia. Video microscopy showed the importance of cytoplasmic flow in colony development with continuous forward flow to support hyphal morphogenesis and reverse flow to support an extended period of conidiogenesis. Our results indicate that the circadian system of Neurospora can be expressed at the level of cellular mass formation, not just as the developmental conidiation rhythm.     

Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.     

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.     

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.     

Temperature-sensitive and circadian oscillators of Neurospora crassa share components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.     

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.     

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.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

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.     

Isolation and characterization of cadmium-resistant mutants of Neurospora crassa

This study identified and characterized four cadmium-resistant mutants of Neurospora crassa. One of these mutants maps to linkage group II and the other three map to linkage group VII, whereas a naturally occurring resistant trait in a strain from Japan resides at a distinct but unmapped locus. Transport of cadmium into Neurospora cells occurs by more than a single uptake system and involves both energy-dependent and -independent components. The resistant mutants transport cadmium in the same manner as does the cadmium-sensitive wild-type strain. Cadmium resistance in these mutants does not appear to result from an increase in cytosolic heat-stable cadmium-binding proteins. Cadmium does not induce the typical heat-shock response in conidia. Under various growth conditions, each of the mutants exhibited morphological alterations, possibly involving the cell wall or plasma membrane.