Complementation Analysis of Linked Circadian Clock Mutants of NEUROSPORA CRASSA

A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq⁺ allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.     

Circadian Rhythms in NEUROSPORA CRASSA: Interactions between Clock Mutations

Mutations at four loci in Neurospora crassa that alter the period of the circadian rhythm have been used to construct a series of double mutant strains in order to detect interactions between these mutations. Strains carrying mutations at three of these loci have altered periods on minimal media: prd-1, several alleles at the olir (oligomycin resistance) locus and four alleles at the frq locus. A mutation at the fourth locus, cel, which results in a defect in fatty acid synthesis, also leads to lengthening of the period when the medium is supplemented with linoleic acid (18:2). The cel mutation was crossed into strains carrying the frq, prd-1 and olir mutations, and the periods of the double mutant strains with and without 18:2 supplementation were determined. In addition, data from the literature for other combinations of loci and/or chemical effects on the period have been reanalyzed.--It was found that both prd-1 and olir are epistatic to the effects of 18:2 on cel; in the series of cel frq double mutant strains, the period-lengthening effect of 18:2 is inversely proportional to the period of the frq parent, indicating an interaction between frq and cel; period effects reported in the literature can be described as changes by a fixed ratio or percentage of the period rather than by a fixed number of hours, and the data, therefore, can support a multiplicative as well as an additive model.--Several biochemical interpretations of these interactions are discussed, based on simple chemical kinetics, enzyme inhibition kinetics and the control of flux through metabolic pathways.     

Isolation and Characterization of Light-Insensitive Mutants of NEUROSPORA CRASSA

As part of a genetic analysis of blue light photoreception in Neurospora, three mutants were isolated that do not exhibit photosuppression of circadian conidiation, i.e., they show periodic conidiation in constant light. The mutations have been given the designations lis-1, lis-2 and lis-3 ("light insensitive"). The three mutations segregate as single nuclear genes, are nonallelic and are recessive to wild type in heterokaryon tests. The linkage groups of the mutations are as follows: lis-1, I; lis-2, VI; and lis-3, V. The light -insensitive phenotype of the mutants is restricted to the photosuppression response; other responses such as photoinduced phase shifting of the conidiation rhythm and photoinduced carotenogenesis are not altered. The physiological or biochemical defects of the mutants have not been established, but they are not similar to previous reported cases (i.e., rib and poky) in which a reduction in light sensitivity has been observed.     

Isolation and Classification of Extranuclear Mutants of NEUROSPORA CRASSA

Four extranuclear mutants, [exn-1], [exn-2],[exn-4], and [stp-C], were obtained from N-methyl-N'-nitro-N-nitrosoguanidine-treated conidia and mycelium of Neurospora crassa. The three exn mutants grow with a pronounced lag from conidia and ascospores and are female fertile, whereas [stp-C] has a stop-start growth phenotype and is female sterile. The mitochondria from all four mutants are deficient in cytochromes a+a(3) and b, but contain an excess of cytochrome c. On the basis of growth and fertility, nuclear suppressors and complementation in heteroplasmons, 16 of the extranuclear mutants now available in Neurospora can be divided into three groups. Group I consists of 8 female-fertile variants with both poky-like growth and cytochrome defects. Their slow growth is suppressed by the nuclear factor, f, but not by a second nuclear suppressor, su-1([mi-3]). They complement with group III mutants in mixed cytoplasmons. Group II is represented by a single variant, [mi-3]. It is phenotypically modified by the su-1([mi-3]) factor, but not by f. Its unique cytochrome spectrum shows a deficiency of cytochrome a, but c and b are present. It complements in heteroplasmons with group I and III mutants. Group III included 7 female-sterile variants with stopper growth phenotypes and the same cytochrome defects as group I. Group III mutants complement both with group I and II isolates, but they are unaffected by either f or su-1.     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

Isolation and Characterization of Temperature-Sensitive Respiratory Mutants of NEUROSPORA CRASSA

Filtration-enrichment and inositol-less death methods of mutant isolation, coupled with a screen for cyanide-insensitive respiration, proved to be highly efficient methods for isolating temperature-sensitive (ts) nuclear Neurospora mutants having defective respiration. Eighteen different ts respiratory mutants have been isolated. Most of them are pleiotropic and defective in one or more of the following phenotypes: cytochrome aa3, b, and c (individual or multiple defects); oligomycin inhibition of ATPase activity; respiration and its inhibition by KCN and salicyl hydroxamic acid; and growth rates in liquid and solid media at 25 degrees and 38 degrees. Among these mutants are the first cytochrome c mutant of Neurospora and an extranuclear ts ATPase mutant. An added bonus was the fact that over half of the mutants were affected either in ribosome assembly or in protein synthesis in the mitochondrion. We have yet to find any mutants completely lacking activities associated with the respiratory chain. However, the wide spectrum of mutants isolated here, along with those currently available, constitutes a considerable resource for investigating respiration in obligate aerobes.     

The Isolation of Mms- and Histidine-Sensitive Mutants in NEUROSPORA CRASSA

A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two others were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25° than at 37°. In contrast, mycelial growth is more resistant to MMS at 37°. The possibility of qualitatively different repair processes at these two temperatures is discussed.     

A Recessive Circadian Clock Mutation at the frq Locus of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa, the most distinctive characteristic of which is the complete loss of temperature compensation of its period length, maps to the frq locus where seven other clock mutants have previously been mapped. This mutant, designated frq-9, is recessive to the wild-type allele and to each of the other frq mutants; thus, it differs from the other mutants, which show incomplete dominance to wild type and to each other. Complementation analysis suggests either that the frq locus is a single gene or that frq-9 is a deletion that overlaps adjacent genes. Preliminary efforts at fine structure mapping have indicated that recombination between certain pairs of frq mutations is less than 0.005%, a distance consistent with the locus being a single gene. The recessive nature of frq-9, coupled with complete loss of temperature compensation, suggests that this mutant may represent the null phenotype of the locus and that the frq gene is involved in the temperature compensation mechanism of the clock.--Genetic mapping studies have placed the frq locus on linkage group VIIR, midway between oli (oligomycin resistance) and for (formate auxotrophy), about 2 map units from each, and clearly indicate that frq and oli are separate genes.     

Genetic and Physiological Characteristics of a Slow-Growing Circadian Clock Mutant of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa with a period length of about 25.8 hours (4 hr longer than wild type) has been isolated after mutagenesis of the band strain. This mutant, called frq-5, segregates as a single nuclear gene, maps near the centromere on linkage group III, and is unlinked to four previously described clock mutants clustered on linkage group VII R (Feldman and Hoyle 1973, 1976). frq-5 differs from the other clock mutants in at least two other respects: (1) it is recessive in heterokaryons, and (2) it grows at about 60% the rate of the parent band strain on both minimal and complete media. Double mutants between frq-5 and each of the other clock mutants show additivity of period length--two long period mutants produce a double mutant whose period length is longer than either of the two single mutants, while a long and a short period double mutant has an intermediate period length. Although slow growth and long periodicity of frq-5 have segregated together among more than 300 progeny, slow growth per se is not responsible for the long period, since all the double mutants have the slow growth characteristic of frq-5, but have period lengths both shorter and longer than wild type.     

Selection of Extranuclear Mutants of NEUROSPORA CRASSA

A procedure is described that produce primarily extranuclear mutants, of Neurospora carassa. An analysis of five mutants. [cni-3], [rsp-2], [rsp-3], E1RSP-4], IS PRESENTED. All five mutants segregate in an extranuclear manner. They can be assorted into two classes based on their respiratory properties: (1) those with cyanide-insensitive respiration (cni); (2) those with slow respiration (rsp). All of the mutants are female sterile. The respiratory trait can be placed in different nuclear backgrounds by heterokarytoic transfer. The abnormal respiratory traits are observed in mitochondria isloated from the mutants and it is likely that the mutations are in mitochondrial DNA.     

The Isolation and Characterization of Mutants Defective in Nitrate Assimilation in NEUROSPORA CRASSA

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing co-factor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit-8 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.     

The frq Locus in NEUROSPORA CRASSA: A Key Element in Circadian Clock Organization

Four new circadian clock mutants of Neurospora crassa have been isolated that alter the period length of the circadian conidiation rhythm. Three of these are at the frq locus on linkage group VIIR, where four other clock mutants are located. In contrast to wild type, which has a period length of 21.6 hr, frq-6 has a period length of 19 hr, while frq-7 and frq-8 have period lengths of 29 hr and represent the largest effects of any single gene mutants on circadian periodicity. Thus, seven mutants have now been isolated that map to the frq locus, with period lengths ranging from 16.5 to 29 hr, and each mutant alters clock periodicity by an integral multiple of 2.5 hr. In addition, all frq mutants show incomplete dominance in heterokaryons. The large percentage of clock mutants that map to this locus, coupled with their unique properties, suggests that the frq locus plays an important role in clock organization.—The fourth mutant, designated chrono (chr), has a period length of 23.5 hr, shows incomplete dominance and is unlinked to either of the previously identified clock loci, frq or prd (formerly called frq-5). Double mutants between various combinations of clock mutants show additive effects and indicate no significant gene interaction among mutants at these three loci.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Isolation of Mutants Deficient in the Repressible Alkaline Phosphatase

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.