System analysis of Phycomyces light-growth response. Photoreceptor and hypertropic mutants.

The light-growth responses of Phycomyces behavioral mutants, defective in genes madB, madC, and madH, were studied with the sum-of-sinusoids method of system identification. Modified phototropic action spectra of these mutants have indicated that they have altered photoreceptors (P. Galland and E.D. Lipson, 1985, Photochem. Photobiol. 41:331). In the two preceding papers, a kinetic model of the light-growth response system was developed and applied to wild-type frequency kernels at several wavelengths and temperatures. The present mutant studies were conducted at wavelength 477 nm. The log-mean intensity was 6 X 10(-2)W m-2 for the madB and madC night-blind mutants, and 10(-4)W m-2 for the madH hypertropic mutant. The prolonged light-growth responses of the madB and madC mutants are reflected in the reduced dynamic order of their frequency kernels. The linear response of the hypertropic mutant is essentially normal, but its nonlinear behavior shows modified dynamics. The behavior of these mutants can be accounted for by suitable modifications of the parametric model of the system. These modifications together support the hypothesis that an integrated complex mediates sensory transduction in the light responses and other responses of the sporangiophore.     

System analysis of Phycomyces light-growth response: madC, madG, and madH mutants.

The light-growth response of Phycomyces has been studied further with the sum-of-sinusoids method in the framework of the Wiener theory of nonlinear system identification. The response was treated as a black box with the logarithm of light intensity as the input and elongation rate as the output. The nonlinear input-output relation of the light-growth response can be represented mathematically by a set of weighting functions called kernels, which appear in the Wiener intergral series. The linear (first-order) kernels of wild type, and of single and double mutants affected in genes madA to madG were determined previously with Gaussian white noise test stimuli, and were used to investigate the interactions among the products of these genes (R.C. Poe, P. Pratap, and E.D. Lipson. 1986. Biol. Cybern. 55:105.). We have used the more precise sum-of-sinusoids method to extend the interaction studies, including both the first- and second-order kernels. Specifically, we have investigated interactions of the madH ("hypertropic") gene product with the madC ("night blind") and madG ("stiff") gene products. Experiments were performed on the Phycomyces tracking machine. The log-mean intensity of the stimulus was 6 x 10(-2) W m-2 and the wavelength was 477 nm. The first- and second-order kernels were analyzed in terms of nonlinear kinetic models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmission and expression of mutations to nalidixic acid resistance among products of protoplast fusion crosses of Candida albicans

Earlier studies suggested that heritable resistance to nalidixic acid (Nal) induced in the asexual, pathogenic yeast Candida albicans by growth on Nal results from mitochondrial mutation. To determine conclusively whether mutations to Nal resistance are cytoplasmic or nuclear, several stable Nal-resistant (Nalr) mutants exhibiting distinctive differences in degrees of Nal resistance were obtained from each of two doubly auxotrophic strains (Ade-, Thr- and Arg-, His-), both derived from the same wild-type stock. Inheritance of Nal resistance was then assessed in a series of protoplast fusion crosses between complementing auxotrophs. The initial, intact cellular products of a fusion cross are prototrophic heterokaryons which frequently assort single parental nuclei into monokaryotic blastospores containing biparental cytoplasms. Occasional karyogamy within heterokaryons also yields prototrophic hybrid monokaryons which can undergo recombinations for chromosomal markers through spontaneous or induced mitotic crossing-over. Segregation and expression of Nal resistance among non-hybrid, parental-type monokaryons from Nalr X Nals heterokaryons showed that Nalr mutations are nuclear and that their expressions are not noticeably affected by admixture of cytoplasms of sensitive and resistant parental strains. Analyses of heterokaryons and hybrid monokaryons from Nalr X Nals and Nalr X Nalr crosses demonstrated that Nal resistance is recessive to sensitivity, and that independent Nalr mutations arise at one gene in the Ade-, Thr- strain and at a separate, complementing single gene in the Arg-, His- strain. Prior work demonstrated that induction of Nalr mutations in wild-type C. albicans depends profoundly on the (i) carbon and nitrogen, (ii) growth temperature, (iii) contact with particular metabolic inhibitors and (iv) division stage of cells during exposure to Nal. The present observations indicate that the character of cellular auxotrophies can determine the genetic loci at which Nalr mutations can be recovered.     

Complementation and noncomplementation in heterokaryons of three unlinked pigment mutants of the fowl.

This study shows that melanocyte heterokaryons formed between cells of the blue and recessive white genotypes complement one another to produce normal pigmentation, while heterokaryons of the blue and pinkeye genotypes fail to complement. The simplest interpretation of these findings is that the blue and recessive white mutations affect different aspects of pigment synthesis so that when both kinds of nuclei exist in the same cytoplasm, they can correct (complement) each other's defect. On the other hand, the blue and pinkeye mutations, although unlinked, apparently affect the same aspect of pigment synthesis so that when both kinds of nuclei are in a common cytoplasm, they cannot correct each other's defect. This suggests that one of these two loci exerts some kind of control, or "regulation," over the other. It has previously been shown that recessive white--pinkeye heterokaryons can complement. Thus, only two heterokaryon complementation groups are evident within the three mutants examined.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Light and dark adaptation in Phycomyces phototropism

Light and dark adaptation of the phototropism of Phycomyces sporangiophores were analyzed in the intensity range of 10(-7)-6 W X m- 2. The experiments were designed to test the validity of the Delbruck- Reichardt model of adaptation (Delbruck, M., and W. Reichardt, 1956, Cellular Mechanisms in Differentiation and Growth, 3-44), and the kinetics were measured by the phototropic delay method. We found that their model describes adequately only changes of the adaptation level after small, relatively short intensity changes. For dark adaptation, we found a biphasic decay with two time constants of b1 = 1-2 min and b2 = 6.5-10 min. The model fails for light adaptation, in which the level of adaptation can overshoot the actual intensity level before it relaxes to the new intensity. The light adaptation kinetics depend critically on the height of the applied pulse as well as the intensity range. Both these features are incompatible with the Delbruck-Reichardt model and indicate that light and dark adaptation are regulated by different mechanisms. The comparison of the dark adaptation kinetics with the time course of the dark growth response shows that Phycomyces has two adaptation mechanisms: an input adaptation, which operates for the range adjustment, and an output adaptation, which directly modulates the growth response. The analysis of four different types of behavioral mutants permitted a partial genetic dissection of the adaptation mechanism. The hypertropic strain L82 and mutants with defects in the madA gene have qualitatively the same adaptation behavior as the wild type; however, the adaptation constants are altered in these strains. Mutation of the madB gene leads to loss of the fast component of the dark adaptation kinetics and to overshooting of the light adaptation under conditions where the wild type does not overshoot. Another mutant with a defect in the madC gene shows abnormal behavior after steps up in light intensity. Since the madB and madC mutants have been associated with the receptor pigment, we infer that at least part of the adaptation process is mediated by the receptor pigment.     

Behavioral Mutants of DROSOPHILA MELANOGASTER. II. Behavioral Analysis and Focus Mapping

Several simple tests have been applied to study the behavior and performance of mutants of Drosophila melanogaster isolated in the preceding study (Homyk and Sheppard 1977). The tests showed that many mutants have specific behavioral abnormalities and that most mutants can easily be distinguished from an Oregon-R control on the basis of their behavioral phenotypes. Mutants representing six genes hop poorly and are unable to initiate wing oscillation when tethered. Mutations in four genes reduce the level of spontaneous motor activity of flies and increase the excitability threshold necessary to induce high activity motor functions such as running and flying. The latter mutants are referred to as hypoactive. Another class, stress-sensitive, including mutations in three genes, are reversibly paralyzed by mechanical shock. Mosaic analyses suggest that six mutations affect muscular tissue and two others affect neural tissue. It is also shown that tan mutants fail to retract their forelegs during flight and that the focus responsible for this behavioral phenotype is the compound eye. Specific behavioral abnormalities of several mutants are discussed in conjunction with previous studies from many laboratories concerning the participation of neural, sensory and muscular elements producing behavior in normal (nonmutant) insects. Such considerations are an essential prelude to anatomical and physiological studies of the mutants in Drosophila.     

Genetic Analysis of Defecation in Caenorhabditis Elegans

Defecation in the nematode Caenorhabditis elegans is achieved by a cyclical stereotyped motor program. The first step in each cycle is contraction of a set of posterior body muscles (pBoc), followed by contraction of a set of anterior body muscles (aBoc), and finally contraction of specialized anal muscles that open the anus and expel intestinal contents (Exp). By testing existing behavioral mutants and screening for new mutants that become constipated due to defects in defecation, I have identified 18 genes that are involved in defecation. Mutations in 16 of these genes affect specific parts of the motor program: mutations in two genes specifically affect the pBoc step; mutations in four genes affect the aBoc step; mutations in four genes affect the Exp step; and mutations in six genes affect both aBoc and Exp. Mutations in two other genes affect the defecation cycle period but have a normal motor program. Sensory inputs that regulate the cycle timing in the wild type are also described. On the basis of the phenotypes of the defecation mutants and of double mutants, I suggest a formal genetic pathway for the control of the defecation motor program.     

Meiosis in NEUROSPORA CRASSA. I. the Isolation of Recessive Mutants Defective in the Production of Viable Ascospores

A scheme has been devised for efficient isolation of recessive meiotic mutants of Neurospora crassa. These mutants were detected by their reduced fertility or by the abortion of ascospores. Their isolation involved the selection and screening of the strains arising from ascospores disomic (n + 1) for linkage group I (LG I), which bears the mating-type locus. These strains are self-fertile heterokaryons that contain two types of haploid nuclei of opposite mating types (A + a). Selfings of these strains are homozygous for genes on all linkage groups except LGI and therefore allow the expression of recessive mutants with an altered sexual cycle. Using this selection procedure, three classes of mutants were detected. In one class, mutants had an early block in perithecial development (class I), and in another mutants had altered perithecia, but apparently unaltered fertility (class III). No recessive mutants were observed and all mutants tested (eight of class I and two of class III) were expressed only when used as the maternal parent. A third mutant class displayed normal production of perithecia, but defective formation of asci (class IIA), or black ascospores (class IIB). Four of 13 class IIA mutants were analyzed, and two of them [asc(DL131) and asc (DL400)] were definitely recessive analysis of 10 of 13 class IIB mutants disclosed six recessive, mutually complementing mutants: ase(DL95), asc(DL243), asc(DL711), asc(DL879), asc(DL917m) and asc(DL961). Mutants asc(DL95), asc(DL243) and the previously studied mei-1 mutant (Smith 1975) complemented one another in crosses, but did not recombine. These may be alleles of the same gene, or they may comprise a gene cluster.     

Parasexual genetic analysis of aggregation-deficient mutants of Dictyostelium discoideum.

One hundred and thirty-nine independent, nitrosoguanidine-induced mutants blocked early in development were isolated in two haploid strains of D. discoideum. Forty of these developmental mutants were completely aggregation-deficient on bacterial lawns (Class I mutants) and these mutants were selected for parasexual genetic analysis. By fusing the Class I mutants with developmentally-competent strains the developmental mutations in 39 of these mutants were shown to be recessive; the remaining mutation appeared to be partially dominant. Complementation analysis of the developmental mutations in the Class I strains identified 5 complementation groups. Statistical analysis of the complementation data suggests that there are approximately 40 genes in this organism which will completely block aggregation when mutated and perhaps as many as 150 genes involved in some aspect of the aggregation process. Linkage analysis of 18 Class I developmental mutations revealed that 10 of these mutations map in linkage group II at a minimum of 5 loci.     

UV-induced recessive lethals in uvs strains of Neurospora which are deficient in UV mutagenesis

The frequencies of spontaneous and UV-induced recessive lethal mutations were compared for UV-sensitive and wild-type heterokaryons of Neurospora crassa. These heterokaryons were homokaryotic either for one of two alleles of uvs-3, or for uvs-6 or uvs+. For uvs-3, which is known to have mutator effects, spontaneous recessive lethals were found to be 4-6 times more frequent than observed in uvs+. After correction for clonal distribution of spontaneous mutants, an observed 2-fold increase for uvs-6 was not statistically significant and may have been due to chance occurrence of a few large clones of mutants. Treatment with low doses of UV (50-200 J/m2) produced very similar overall rates of increase for recessive lethals in uvs and uvs+ heterokaryons. This means, that in contrast to results obtained when mutation to ad-3 was measured, both uvs-3 alleles showed highly significant increases for recessive lethals when treated with UV. It is proposed that certain types of UV damage may be processed into recessive lethal mutations by an alternate mechanism from that responsible for viable mutations.     

Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora.

Summary The relative frequencies of heterokaryons and the two kinds of homokaryons have been scored among colonies from conidia harvested from a heterokaryon and treated with UV, in order to determine which kinds of lethal mutations were induced. Recessive lethal mutations were scored directly. The pattern of surviving types indicated that recessive lethals and mitotic lethals (events destroying whole nuclei) occurred with similar frequencies. But the absolute frequency of these mutations was not sufficient to account for the observed kill, suggesting that dominant lethals and/or cytoplasmic lethals were also induced at a similar rate.     

Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.     

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.     

Growth rate fluctuations in phycomyces sporangiophores

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input (“night-blind”) or output (“stiff” and “hypertropic”) of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.     

The enhancer of split locus and neurogenesis in Drosophila melanogaster

Enhancer of split (E(spl)) is one of a group of so-called neurogenic genes of Drosophila. We describe two different types of E(spl) alleles, dominant and recessive, which exert opposite effects on both central and peripheral nervous system development. The only extant dominant allele determines a reduction in the number of central neurons and peripheral sensilla; this phenotype is not reduced by a normal complement of wild-type alleles. Since animals carrying a triploidy for the wild-type locus develop similar defects, the dominant allele is probably the result of a gain-of-function mutation. Several recessive alleles, obtained as revertants of the dominant allele, are loss-of-function mutations and determine considerable neural hyperplasia. The present evidence suggests that neural defects of E(spl) mutants are due to defective segregation of neural and epidermal lineages, leading to neural commitment of less or of more cells than in the wild type, depending upon whether the animals carry the dominant or any of the recessive alleles, respectively. Therefore, E(spl) formally behaves as a gene switching between neural and epidermal pathways.     

An enzyme complex for the dehydrogenation of phytoene in Phycomyces.

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.     

MUTATIONS AFFECTING HETEROKARYOSIS IN SCHIZOPHYLLUM COMMUNE

Mutations that affect the basic characteristics of heterokaryons of S. commune occur spontaneously and are preferentially selected in the common-A heterokaryon and in its homokaryotic mimics, strains carrying a mutated B factor or strains disomic for heteroallelic B factors. Nine independent mutations were compared: all segregate independently of A and B incompatibility factors, and at least 3 distinct loci, of which 2 are linked, are involved. None of the mutations is phenotypically expressed in the homokaryon or in the common-AB heterokaryon. All 9 mutations increase vegetative vigor of the common-A the effects of all the mutations are additive in both heteroallelic and homoallelic combinations. At least 1 type-II mutation also affects nuclear distribution in common-B heterokaryons. Type-II mutations appear to reduce common-A, common-B, and compatible heterokaryons to a single type unlike any of the normal heterokaryons. Pseudoclamping often persists for extended periods in modified homokaryons isolated from modified heterokaryons. Several cases of somatic recombination have been observed among components of modified heterokaryons.     

Constitutive mutants in the yeast pheromone response: ordered function of the gene products

The alpha factor pheromone inhibits the division of yeast a cells. A general method was developed for isolating mutants that exhibit constitutive activation of the pheromone response pathway. A dominant allele of the STE4 locus was recovered in addition to recessive mutations in the SCG1 gene. SCG1 and STE4 are known to encode G alpha and G beta homologs, respectively. Analysis of double mutants suggests that the STE4 gene product functions after the SCG1 product but before the STE5 product.