Isolation and Characterization of a Temperature-Sensitive Circadian Clock Mutant of Neurospora Crassa

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period-6 (prd-6), has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21°) the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21°) the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperature-sensitive aspect of the phenotype of the prd-6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal prd-2(+) protein is required for low temperature to ``rescue' the prd-6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock.     

Epistatic and synergistic interactions between circadian clock mutations in Neurospora crassa

We identified a series of epistatic and synergistic interactions among the circadian clock mutations of Neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. The period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. The prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is considerably longer than predicted. In addition, the prd-2 prd-3 double mutant strain also exhibits overcompensation to changes in ambient temperature, suggesting a role in the temperature compensation machinery of the clock. The prd-2, prd-3, and prd-6 mutations also show significant interactions with the frq(7) long-period mutation. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in both the timing and temperature compensation mechanisms of the circadian clock and may interact with the FRQ protein.     

Circadian rhythms in Neurospora crassa: clock mutant effects in the absence of a frq-based oscillator

In Neurospora, the circadian rhythm is expressed as rhythmic conidiation driven by a feedback loop involving the protein products of frq (frequency), wc-1 (white collar-1), and wc-2, known as the frq/wc (FWC) oscillator. Although strains carrying null mutations such as frq(10) or wc-2Delta lack a functional FWC oscillator and do not show a rhythm under most conditions, a rhythm can be observed in them by the addition of geraniol or farnesol to the media. Employing this altered media as an assay, the effect of other clock mutations in a frq(10)- or wc-2Delta-null background can be measured. It was found that the existing clock mutations fall into three classes: (1) those, such as prd-3 or prd-4 or frq(1), that showed no effect in a clock null background; (2) those, such as prd-1 or prd-2 or prd-6, that did have a measurable effect in the frq(10) background; and (3) those, such as the new mutation ult, that suppressed the frq(10) or wc-2Delta effect, i.e., geraniol/farnesol was not required for a visible rhythm. This classification suggests that some of the known clock mutations are part of a broader multioscillator system.     

Circadian rhythms in Neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition

The fatty acid compositions of the phospholipids of Neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. In liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. By late log phase, fatty acid composition was essentially constant. The phospholipid fatty acid compositions of bd strains containing mutations at the frq (frequency) and chr (chrono) loci were indistinguishable from that of the bd strain under the conditions used. However, a bd strain containing a mutation at the prd-1 (period) locus, as well as prd-1 segregants from a cross of this strain to a bd strain, had altered patterns of growth-dependent fatty acid composition; linoleic and linolenic acid contents changed more slow than in the bd strain and continued to change throughout growth. In addition, the fatty acid composition of a bd prd-1 strain on solid medium differed from that of the bd strain. It is proposed that the prd-1 mutation leads to altered membrane homeostasis, which in turn affects circadian rhythmicity because some or all components of the rhythm-generating system are membrane-localized.     

Mutations that affect circadian rhythms in Neurospora crassa can alter the reduction of cytochromes by blue light

We have examined membrane fractions from mutant strains of Neurospora crassa that have altered responses to blue light or have altered circadian rhythms. Using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. Three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. The poky strain was found to have high concentrations of photoreducible cytochrome c. After removal of this cytochrome, however, the photoreducible cytochromes in the plasma membrane and endoplasmic reticulum were also similar to those of the wild type. The most significant differences were found in strains mutated at the frq locus, which affects circadian rhythms. In the frq-9 strain, the cytochrome in the endoplasmic reticulum was not detectably reduced by blue light. The frq-1 mutation caused a significant shift in the spectrum of blue-light-reduced cytochrome in the endoplasmic reticulum.     

Gene dosage effects on the synthesis of maltase in yeast.

Inbred strains of Saccharomyces cerevisiae carrying MAL1, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MAL1, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects of the differential rate of maltase synthesis (delta enzyme activity/delta growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MAL1 and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MAL1 and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low genes dosages.     

Loss of temperature compensation of circadian period length in the frq-9 mutant of Neurospora crassa

A new circadian clock mutant of Neurospora crassa has been isolated, whose most distinctive characteristic is the complete loss of temperature compensation of its period length. The Q10 of the period length was found to be equal to about 2 in the temperature range from 18 degrees to 30 degrees C. The period length was also found to be dependent on the composition of the medium, including the nature and concentration of both the carbon source and the nitrogen source. Although the rate of the clock and the growth rate were directly related when affected by varying the temperature, they were inversely related when altered by changing the composition of the medium. Therefore, the mutation has not simply coupled clock rate to growth rate in this strain. The mutation maps to the frq locus, where seven other clock mutations previously studied also map. Therefore, this mutant has been called frq-9. Since several of the other frq mutants show partial loss in temperature compensation, it is suggested that the frq gene or its product is closely related to the temperature compensation mechanism of the circadian clock of Neurospora.     

A two-locus mutation—Selection model and some of its evolutionary implications

The deterministic properties of a two-locus model with mutation and selection have been investigated. The mutation process is unidirectional, and the model is so constructed that the genetic variation at one locus is selectively neutral in the absence of a mutant allele at the other locus. All genotypes with three or four mutant alleles are deleterious, while the double heterozygotes may have the same fitness as the standard genotype. If one of the mutant alleles becomes fixed in the population, then the other locus will show a regular one-locus mutation-selection balance. Such a boundary equilibrium may be unstable or stable in the full two-locus setting. In the symmetric case, which is analyzed in details, the population will either go to one of the two boundary equilibria, or to a fully polymorphic equilibrium at which both the mutant alleles are rare. The origin of reproductive separation between two populations via the fixation of complementary deleterious mutants at different loci, and the fixation of nonfunctional alleles at duplicated loci, are two biological processes which both can be studied with the present model. In the last part of the paper we show how the results from the deterministic analysis can be used to predict how different factors will influence the rates of evolution in these systems.     

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.     

The a locus governs cytoduction in Ustilago maydis.

We have developed a cytoduction assay to measure cell fusion quantitatively in the basidiomycete corn smut fungus Ustilago maydis. This assay employs a mutation conferring resistance to oligomycin that exhibits non-Mendelian inheritance and presumably affects the mitochondrial genome. After auxotrophic olir cells are mixed with prototrophic olis cells, prototrophic olir cells can be detected at a significant frequency after several hours of incubation, reaching a maximum of 10% of the total prototrophs in the mixture after 18 h. We demonstrate that this cell fusion event occurs only if the mating partners have different alleles of the a mating-type locus and is not influenced by the b locus. These studies support the view that the a locus but not the b locus controls establishment of the filamentous, pathogenic state.     

Analysis of the spectra of mutations and polymorphic loci of cystic fibrosis transmembrane conductance regulator in the population of Bashkortostan

The spectra of mutations and polymorphic loci of the gene of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in 60 cystic fibrosis (CF) families from Bashkortostan. Mutations delF508, 394delTT, CFTRdele2,3(21 kb), R334W, and S1196X (33.3, 3.3, 1.7, 0.8, and 0.8%, respectively) were identified. The frequencies of tandem tetranucleotide repeat (TTR) alleles were determined for locus IVS6a-GATT of intron 6 of the CFTR gene and two extragenic loci flanking the CFTR gene, D7S23 and MET (probes CS.7 and MetH) in mutant and normal chromosomes. Allelic and haplotypic associations of these loci with the mutations found were estimated. An absolute linkage between the 6TTR allele of locus IVS6a-GATT and the delF508 mutation was ascertained. A considerable linkage disequilibrium between the delF508 mutation and the C2 allele of locus D7S23 and between this mutation and the A1 allele of locus MET was found. Most of the other mutant chromosomes carried marker alleles 7TTR, C1, and A2. It was demonstrated that 67% of CF chromosomes carrying delF508 had haplotype 6-2-1 for loci IVS6a-GATT/D7S23/MET, respectively. The frequency distribution of haplotypes in CF chromosomes without delF508 had a high variance and did not differ significantly from the distribution in normal chromosomes (chi 2 = 9.415; p > 0.05).     

Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system

The CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5 min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95 min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild-type and corA mutations. Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co(2+)-resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild-type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficient to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild-type allele of at least one of the corB, corC or corD loci.     

A Fourth Escherichia Coli Gene System with the Potential to Evolve β-Glucoside Utilization

Escherichia coli K12 is being used to study the potential for adaptive evolution that is present in the genome of a single organism. Wild-type E. coli K12 do not utilize any of the beta-glucoside sugars arbutin, salicin or cellobiose. It has been shown that mutations at three cryptic loci allow utilization of these sugars. Mutations in the bgl operon allow inducible growth on arbutin and salicin while cel mutations allow constitutive utilization of cellobiose as well as arbutin and salicin. Mutations in a third cryptic locus, arbT, allow the transport of arbutin. A salicin+ arbutin+ cellobiose+ mutant has been isolated from a strain which is deleted for the both the bgl and cel operons. Because the mutant utilized salicin and cellobiose as well as arbutin, it is unlikely it is the result of a mutation in arbT. A second step mutant exhibited enhanced growth on salicin and a third step mutant showed better growth on cellobiose. A fourfold level of induction in response to arbutin and a twofold level of induction in response to salicin was observed when these mutants were assayed on the artificial substrate p-nitrophenyl-beta-D-glucoside. Although growth on cellobiose minimal medium can be detected after prolonged periods of time, these strains are severely inhibited by cellobiose in liquid medium. This system has been cloned and does not hybridize to either bgl or cel specific probes. We have designated this gene system the sac locus. The sac locus is a fourth set of genes with the potential for evolving to provide beta-glucoside utilization.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

Initiation of sporulation in Saccharomyces cerevisiae. Mutations causing derepressed sporulation and G1 arrest in the cell division cycle

Mutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 degrees C and 30 degrees C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 degrees C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13.9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.     

Expression of the ROAM mutations in Saccharomyces cerevisiae: involvement of trans-acting regulatory elements and relation with the Ty1 transcription

The regulatory mutations in Saccharomyces cerevisiae designated cargA + Oh, cargB + Oh, and durOh are alterations in the control regions of the respective structural genes. The alteration causing the cargA + Oh mutation has been shown to be an insertion of a Ty1 element in the 5' noncoding region of the CAR1 ( cargA ) locus. All three mutations cause overproduction of their corresponding gene products and belong to the ROAM family of mutations (Regulated Overproducing Allele responding to Mating signals) in yeast. The amount of overproduction in ROAM mutants is determined, at least in part, by signals that control mating functions in yeast. We report the identification of two genetic loci that regulate Oh mutant gene expression but that do not affect mating ability. These loci are defined by the recessive roc mutations ( ROAM mutation control) that reduce the amount of overproduction caused by the cargA + Oh, cargB + Oh, and durOh mutations. RNAs homologous to CAR1 ( cargA ), DUR1 ,2 and Ty1 DNA probes were analyzed by the Northern hybridization technique. In comparison with wild-type strains, cargA + Oh and durOh mutant strains grown on ammonia medium contain increased amounts of CAR1 and DUR1 ,2 RNA. This RNA overproduction is diminished in MATa/MAT alpha diploid strains as well as in haploid strains that also carry the ste7 mutation which prevents mating or that carry either of the roc1 or roc2 mutant alleles. The amount of RNA homologous to Ty1 DNA is also reduced in ste7 , roc1 , and roc2 mutant strains. This reduction is not observed in a strain with the ste5 mutation, which prevents mating but has no effect on overproduction of ROAM mutant gene products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coevolution of self-fertilization and inbreeding depression. III. Homozygous lethal mutations at multiple loci.

We study the evolution of the rate of self-fertilization in response to deleterious mutations at multiple loci. Although partial selfing induces associations among loci even in the absence of linkage, associations among mutations at different loci are of a smaller order of magnitude than the mutation rate. Genotypes that carry homozygous lethal mutations in heterozygous form at i loci occur in frequencies of the order (Ti) mu i, in which T denotes the number of viability loci and mu the mutation rate. While associations between mutations at different loci remain small even under inbreeding, each viability locus develops an association with the modifier of the rate of self-fertilization that substantially affects the evolution of the breeding system. Positive associations between enhancers of selfing and haplotypes carrying multiple wild-type alleles and positive associations in heterozygosity between the modifier locus and the viability loci promote evolutionary increases in the rate of self-fertilization.     

Hitchhiking and associative overdominance at a microsatellite locus

The possible effects of a selected locus on a closely linked microsatellite locus are discussed and analyzed in terms of coalescent theory and models of the mutation process. Background selection caused by recurrent deleterious mutations will reduce the variance of allele size at a microsatellite locus. The occasional substitution of advantageous alleles (genetic hitchhiking) will also reduce the variance, but a high mutation rate at a microsatellite locus can restore the variance relatively rapidly. Overdominance at the selected locus will increase the variance at the microsatellite locus and create partitioning of the variation in allele size among gametes carrying one or the other of the overdominant alleles. These results suggest that neutral microsatellite loci can provide indicators of selective processes at closely linked loci.