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.     

An Unstable Allele of the am Locus of NEUROSPORA CRASSA

The mutant strain am126 was isolated, using the direct selection procedure, after nitrous acid mutagenesis. It produced neither measurable NADP-dependent glutamate dehydrogenase (GDH) nor immunologically cross-reacting material. That the am126 strain produced some form of GDH product was shown by the fact that it complemented several other am mutant strains. The GDH formed by complementation between am126 and each of two other am mutants was relatively thermolabile, but could not be distinguished from wild-type GDH formed by electrophoresis in polyacrylamide gels. This, together with the relatively high yield of the complementation enzymes, suggest that the am126 product is a polypeptide chain not grossly abnormal in structure. The spontaneous revertant frequency was between 0.3 and 3 prototrophic revertants per 10(5) live cells. This frequency was at least 40 times greater than that for am19, which had the second highest spontaneous revertant frequency among the mutants tested. Neither meiosis nor mutagenesis increased the revertant frequency, nor did incubation at elevated temperatures lower it. Sixty-eight revertant strains were examined for thermostability of their GHD. All appeared to be identical to wild type. Seven of the revertant strains were also tested for instability with regard to forward mutation to am auxtrophy. None was found to be unstable. Models for the genetic instability of the am126 mutation are discussed.     

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.     

The Diversity of Alleles at the Hsd Locus in Natural Populations of Escherichia Coli

In enteric bacteria three discrete families of type I restriction and modification systems (IA, IB and ID) are encoded by alleles of the serB-linked hsd locus. Probes specific for each of the three familes were used to monitor the distribution of related systems in 37 of the 72 wild-type Escherichia coli strains comprising the ECOR collection. All 25 members of group A in this collection were screened; 12 were probe-positive, nine have hsd genes in the IA family, two in the IB and one in the ID. Twelve strains, representing all groups other than A, were screened; five were probe-positive, one has hsd genes in the IA family, one in the IB and three in the ID. The type ID genes are the first representatives of this family in E. coli, the probe-negative strains could have alternative families of hsd genes. The type IA and IB systems added at least five new specificites to the five already identified in natural isolates of E. coli. The distribution of alleles is inconsistent with the dendrogram of the bacterial strains derived from other criteria. This discrepancy and the dissimilar coding sequences of allelic hsd genes both imply lateral transfer of hsd genes.     

Genetics of Arginine Biosynthesis in NEUROSPORA CRASSA

A large number of arginine-requiring mutants of Neurospora was isolated, using a strain already partially impaired in an enzyme of the pathway. Among the mutants, all previously described loci, except one, were represented, and several new loci were defined and mapped. Four groups of mutants were of particular interest. First, the large group of arg-6 mutants, when tested for intragenic complementation, suggested a bifunctional gene, possibly controlling two steps in ornithine synthesis. This is consistent with the limited enzymic information about this locus. Second, the arg-13 locus was represented by 14 new mutants. All five tested were quite leaky, suggesting that the function controlled by this gene can be carried out to a limited extent spontaneously or by another gene product. Third, a new locus, arg-14, was defined. It controls a step in ornithine syntheses. It lies in a 1 to 2 map-unit interval between arg-2 and pyr-3 on LG IVR, as shown by mapping in relation to translocation breakpoints. Fourth, a second new locus whose mutants render the partial mutation in starting material auxotrophic was defined and mapped near the centromere of LG VIL. These new mutants are unable to derepress enzymes of the pathway and may qualify as regulatory mutants.     

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.     

Genetic Control of Phosphate-Metabolizing Enzymes in NEUROSPORA CRASSA: Relationships among Regulatory Mutations

In Neurospora crassa, the phosphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, preg and nuc-1, pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pcon-c (constitutive) mutations reside in the same cistron. preg-c (constitutive) mutations are epistatic to nuc-2 mutations. nuc-1 (null) mutations are epistatic to all others.     

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F₂ and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.     

Mutations of the a Mating-Type Gene in NEUROSPORA CRASSA

In Neurospora, the mating-type locus controls both mating ( A + a is fertile) and heterokaryosis (A + a is incompatible). The two alleles appear stable: no novel fertility reactions have ever been reported, and attempts to separate fertility and heterokaryon incompatibility functions by recombination have been unsuccessful. In the present approach the locus was studied through a mutational analysis of heterokaryon incompatibility function. A selection system was used that detects vigorous (A + a) heterokaryotic colonies against a background of inhibited growth. Twenty-five mutants of an a strain were produced following mutagenic treatment with UV and NG: 15 were viable as homokaryons and 10 were not. All but one were infertile, but most showed an abortive mating reaction involving the production of barren, well-developed perithecia with A and (surprisingly) a testers. None of the mutants complement each other to restore fertility. Seven mutants have been mapped to the mating-type locus region of chromosome 1. Restoration of fertility was used to detect revertants, and these were found in five out of the eight mutants tested. (A dose response was observed). In four cases incompatibility was fully restored and in one case it was not.—The results suggest two positive actions of the locus when in heterozygous (A/a) combination (the stimulation of some stage of ascus production and the inhibition of vegetative heterokaryosis), and one positive action in homozygous combination (the production of a perithecial inhibitor).     

Isolation of Circadian Clock Mutants of NEUROSPORA CRASSA

Three mutants of Neurospora crassa have been isolated which have altered period lengths of their circadian rhythm of conidiation. The strains, designated "frequency" (frq), were obtained after mutagenesis of the band (bd) strain with N-methyl-N'-nitro-N-nitrosoguanidine. In continuous darkness at 25 degrees bd has a period length of 21.6 +/- 0.5 hours; under the same conditions the period length of frq-1 is 16.5 +/- 0.5 hours; frq-2, 19.3 +/- 0.4 hours; and frq-3, 24.0 +/- 0.4 hours. Each of the mutants segregates as a single nuclear gene. All three mutants appear very tightly linked to each other, but it has not yet been determined whether the mutants are allelic. No major changes in the responses to light and temperature have been observed in any of the mutants. It is suggested that these mutants represent alterations in the basic timing mechanism of the circadian clock of Neurospora.     

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.     

Adaptive Significance of Vegetative Incompatibility in NEUROSPORA CRASSA

Certain features reminiscent of sexuality occur in the vegetative life cycle of some filamentous fungi such as Neurospora crassa. Hyphal fusions can occur between genetically different individuals, thereby endowing the new composite mycelium, a heterokaryon, with some of the advantages of heterozygosity usually associated with diploid organisms. In N. crassa, however, there are a number of incompatibility loci which prevent formation of heterokaryons unless the alleles at the incompatibility loci are identical in the two mycelia. The selection pressures that maintain incompatibility polymorphisms are not known. We suggest here that they are maintained because they prevent a kind of exploitation of heterokaryons by nuclei that are nonadaptive in homokaryons but that enjoy a proliferative advantage over other nuclei in heterokaryons. A mathematical model that abstracts the major features of the vegetative life cycle of Neurosopra crassa has been developed, and the action of selection in this model and various extensions of it is such as to maintain polymorphisms of vegetative incompatibility factors.     

Properties of Two Nuclease Genes in NEUROSPORA CRASSA

Two genes, nuc-1 and nuc-2, of Neurospora crassa which were responsible for the nucleic acid digestion, were located on linkage group 1 and 2, respectively. A temperature-sensitive mutant (B1ts-2) was obtained from a nuc-2 mutant. Nuclease mutants showed a reduced activity of nuclease N(3) which was found to be a complex consisting of nuclease N(3) (') and inhibitor molecule. Nuclease N(3), nuclease N(3) (') and inhibitor were partially purified and estimated to have the approximate molecular weights of 38,000, 12,500 and 25,000 respectively. A nuc-1 mutant produced the nuclease N(3) (') altered in thermolability. A nuc-2 mutant and B1ts-2 produced the inhibitor altered in the capacity to inhibit nuclease N(3) (') activity. The inhibitor prepared from B1ts-2 was more thermostable than that from other strains. From these results, it was suggested that the nuc-1 gene is the structural gene for nuclease N(3) (') and the nuc-2 gene that for the inhibitor. A possible involvement of this enzyme-inhibitor complex in the regulation of nuclease activity and synthesis of other proteins was suggested.