Phototropism mutants of Phycomyces blakesleeanus isolated at low light intensity

Mutants of Phycomyces have played a major role in the analysis of phototropism and other responses. Fifteen new mutants of Phycomyces with abnormal phototropism (genotype mad) have been isolated on the basis of their inability to bend toward dim unilateral blue light (fluence rate 5 × 10⁻⁷ W m⁻²), a protocol different from those employed in previous mutant hunts. One mutant resulted from chemical mutagenesis with ICR-170, and the other 14 were induced with N-methyl-N′-nitro-N-nitro-soguanidine. Seven of the mutants are “night blind”; six have phototropic thresholds intermediate between those of wild type (10⁻⁹ W m⁻²) and madA strains (∼ 10⁻⁴ W m⁻²); and one has a threshold similar to that of night-blind madB and madC mutants. The other eight mutants are “stiff”, with various reductions of tropic responsiveness. Two of them, when compared to previously isolated stiff mutants, show unusually weak responses to light, barriers, and gravity.     

System analysis of Phycomyces light-growth response in single and double night-blind mutants

The sum-of-sinusoids method of nonlinear system identification has been applied to the light-growth response of the Phycomyces sporangiophore. Experiments were performed on the Phycomyces tracking machine with the wild-type strain with single and double mutants affected in genes madA, madB, and madC. The sum-of-sinusoids test stimuli were applied to the logarithm of the light intensity. The log-mean intensity level was 10^-1 Wm^-2 and the wavelength was 477 nm. The system identification results are in the form of first- and second-order frequency kernels, which are related to temporal kernels that appear in the Wiener functional series. The first-order kernels agree well with those obtained previously by the white noise method. In particular, the madA madB and madB madC double mutants show very weak responses. With the superior precision of the sum-of-sinusoids methods, we have achieved sufficient resolution to measure and analyze their second-order kernels. The first- and second-order frequency kernels were interpreted by system analysis methods involving a nonlinear parametric model. In addition a nonparametric hypothesis concerning interactions of gene products was tested. Results from the interaction tests confirm the earlier conclusion that the madB and madC gene products interact. In addition, with the enhanced precision and with the extension to nonlinear analysis, we have found evidence of interaction of the madA gene product with the madB and madC gene products. Thus all three genes appear to have mutual interactions, presumably because of their close physical association in a photoreceptor complex.     

Double mutants of Phycomyces with abnormal phototropism

Summary Seven genes (madA to madG) are known which effect phototropism in Phycomyces. These genes have been partially ordered with respect to the associated stimulus-response pathway. Mutants affected in these genes serve as useful probes of photosensory transduction processes in this model system. To extend and deepen the analysis of the system, we have constructed a family of 21 double mutants in all combinations for the seven mad genes. A set of seven standard alleles was adopted for this work. The double mutants were isolated from crosses between isogenic single-mutant strains of opposite mating type. After a partial physiologic screening of the progeny, the double mutants were identified by complementation tests using single-mutant strains of known genotype. For all but three of the double mutants, the photogeotropism phenotypes were distinct from those of the respective single-mutant parentals. One triple mutant (madA madB madC) was constructed as part of this work. Various applications of the double mutants and the triple mutant are discussed. Recombination analyses were performed on the progeny from seven mad crosses to complete an earlier study. The results establish that all seven mad genes are unlinked.     

Electrophoretic analysis of proteins fromPhycomyces mutants with abnormal tropisms

Certain phototropism mutants ofPhycomyces blakesleeanus show defective bending responses (tropisms) to stimuli besides light, such as gravity, wind, and barriers. These so-called stiff mutants are affected in four genes (madD tomadG). Using two-dimensional gel electrophoresis, we have analyzed polypeptides from microsomal and soluble fractions obtained from the wild type, four single mutants, and six double mutants affected in all pairwise combinations of the four genes. Consistent differences in spot patterns formadE andmadF mutants were found in microsomal fractions but not in soluble fractions. InmadE mutants, two spots designated E1 (52 kDa,pI 6.65) and E2 (50 kDa,pI 6.65) were altered. E1 appeared denser in the wild type than in themadE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control bymadE, perhaps involving posttranslational modification. A protein spot, F1 (53 kDa,pI 6.1), was present on the wild-type gels but absent from all gels formadF mutants. The F1 polypeptide probably represents themadF gene product.This work was supported by research grants to E.D.L. from the National Science Foundation (DMB-8316458 and DMB-8704602) and an equipment grant to C.H.T. from the Syracuse University Senate Research Committee.     

System analysis of Phycomyces light-growth response: Single mutants

The white-noise method of system identification has been applied to the transient light-growth response of a set of seven mutants of Phycomyces with abnormal phototropism, affected in genes madA to madG. The Wiener kernels, which represent the input-output relation of the light-growth response, have been evaluated for each of these mutants and the wild-type strain at a log-mean blue-light intensity of 0.1 W m^-2. Additional experiments were done at 3x10^-4 and 10 W m^-2 on the madA strain C21 and wild-type. In the normal intensity range (0.1 W m^-2) the madA mutant behaves similarly to wild-type, but, at high intensity, the madA response is about twice as strong as that of wild-type. Except for C21 (madA), the first-order kernels of all mutants were smaller than the wild-type kernel. The first-order kernels for C111 (madB) and L15 (madC) show a prolonged time course, and C111 has a longer latency. The kernels for C110 (madE), C316 (madF), and C307 (madG) have a shallow and extended negative phase. For C68 (madD), the latency and time course are shorter than in the wild-type. These features are also reflected in the parameters estimated from fits of the anlytical model introduced in the previous paper to the experimental transfer functions (Fourier transforms of the kernels). The kernel for L15 (madC) is described better by a model that lacks one of the two second-order low-pass filters, because its response kinetics are dynamically of lower order.     

Complementation betweenPhycomyces mutants of mating type (+) with abnormal phototropism

Summary Twelve mutants ofPhycomyces blakesleeanus with defects in sporangiophore phototropism (genotypemad) were obtained from a wild type of the (+) mating type by mutagenesis with nitrosoguanidine. These mutants were tested for genetic complementation against standard (+)mad mutants derived from sexual crosses between the isogenic (+) strain and established (-)mad mutants (Ootaki et al., 1974; Eslava et al., 1976). Heterokaryons for complementation tests were obtained by grafting stage I sporangiophores. The (+) mutants were also investigated for their sensory responses such as photoinduction of sporangiophores and avoidance. The mutants were grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were eleven class 1.2 mutants and one class 2 mutant. Complementation tests revealed that all eleven class 1.2 mutants carry the genemadC and the class 2 mutant carriesmadD. There was no evidence that any were double mutants. These results are consistent with the phenotypic classification and with the complementation results of themad mutants of the (-) mating type.     

Thiostrepton-resistant mutants of Bacillus subtilis: Localization of resistance to the 50S subunit

Summary Sexual crosses were studied between mutants ofPhycomyces blakesleeanus with abnormal phototropism (phenotypemad). Recombination frequencies were determined among five genesmadA tomadE. No clear evidence was found for linkage between any of the genes. Inconsistent results in crosses involvingmadC are attributed to nonisogenicity between the particular strains used. Onemad strain was discovered to be a double mutant. A new gene, tentatively designatedmadG, was segregated from a cross involving that strain.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Light-induced absorbance changes in Phycomyces: evidence for cryptochrome-associated flavosemiquinones

Light-induced absorbance changes (LIACs), which are associated with early photochemical events of blue-light transduction, were detected in growing zones of Phycomyces sporangiophores. The novel LIACs meet all the essential requirements for a spectrophotometric photoreceptor assay which was previously unavailaible for blue-light receptors (cryptochromes). In-vivo absorption spectra of growing zones were derived from reflection spectra which were measured with a novel rapid-scan spectrophotometer. To detect photoreceptor-associated absorbance changes white mutants were employed which lack the interfering bulk pigment β-carotene. Blue and white light, not however red light, induced in these strains absorbance changes near 460–490 and 600–620 nm. The LIACs were absent in light-insensitive mutants with defects in the genes madA, madB and madC. Because these genes affect photosensory adaptation and the blue-light receptor itself, the novel in-vivo LIACs must be associated with photochemical events which occur early in the transduction chain. The spectral characteristics of the LIACs are in accordance with a blue- and red-light absorbing flavosemiquinone which is generated upon light absorption by an oxidized flavin receptor. It is proposed that the flavosemiquinone functions itself as photoreceptor which mediates several red-light responses of Phycomyces. Received: 28 September 1998 / Accepted: 25 November 1998     

Kinetics of photoaccumulation of β-carotene in Phycomyces blakesleeanus

Upon carbon starvation the -carotene content of Phycomyces mycelium grown on minimal agar medium disappears with a time lag of about 90 min and a T_1/2 of 68–75 min. If continuous light is given 2 h after starvation, there is an increase in -carotene content with respect to the dark control. This increase has a time lag of 20–25 min. The fluence rate-response curve of wt is biphasic and two mutants in the gene madA (madA7, madA35) and in the gene madB (madB101, madB104) have higher thresholds than wt; madB mutants are blinder than madA mutants. Only blue light is effective and we suggest that it has an effect solely on the catabolism of -carotene.Abbreviations D dark - L light - wt wild type     

Relationship of photocarotenogenesis to other behavioural and regulatory responses inPhycomyces

The effect of light on carotene accumulation was studied by analyzing the -carotene content of 4--old mycelia continuously exposed to illumination of different intensities. The wild-type, mutants defective in phototropism, mutants defective in carotene regulation, and newpic mutants specifically defective for photocarotenogenesis were examined. The results indicate that photocarotenogenesis depends on a single sensory pathway which shares its earlier steps (governed by genesmadA andmadB) with the sensory pathway for phototropism. It shares its later steps (probably governed by genescarA andpicB) with one of the pathways for carotene regulation, and includes at least one specific step (governed by genepicA) not known to be involved in other responses.     

System analysis of Phycomyces light-growth response: Double mutants

The light-growth response of Phycomyces has been studied with Gaussian white-noise test stimuli for a set of 21 double mutants affected in all pairwise combinations of genes madA to madG; these genes are associated with phototropism, the light-growth response, and other behaviors. The input-output relations of the light-growth responses of these mutants are represented by Wiener kernels in the time domain and transfer functions in the frequency domain. The results have been analyzed comparatively with those in the preceding papers on wild-type and single mutant strains. Two of the double night-blind mutants (combinations AB and BC) have especially weak, but still detectable, responses. To evaluate possible dynamic interactions among the seven mad gene products, each double-mutant transfer function was analyzed jointly with those of the parental single mutants and wild-type. Specifically, a hypothesis of dynamic independence was rejected at the 5% significance level for the following combinations: AD, AE, AG, BC, BD, BE, BF, BG, CD, CE, CF, DE, DG, and EF. A formal pictorial scheme summarizes the dynamic interactions among the mad gene products, according to this test. The high degree of interactions between the input gene products (A, B, and C) and the output gene products (D, E, F, and G) suggest that most or all of the sensory transduction pathway for the light-growth response (and phototropism) is contained in a multimolecular complex.     

Blue Light Reception in Phycomyces: Red Light Sensitization in madC Mutants

Sporangiophores of the zygomycete fungus Phycomyces blakesleeanus are sensitive to near UV and blue light. The quantum effectiveness of yellow and red light is more than 6 orders of magnitude below that of near UV or blue light. Phototropism mutants with a defect in the gene madC are about 10⁶ times less sensitive to blue light than the wild type. These mutants respond, however, to yellow and red light when the long wavelength light is given simultaneously with actinic blue light. In the presence of yellow or red light the photogravitropic threshold of madC mutants is lowered about 100-fold though the yellow and the red light alone are phototropically ineffective. A step-up of the fluence rate of broad-band red light (> 600 nm) from 6 × 10^?3 to 6W m^?2 elicits, in mutant C 148 madC, a transient deceleration of the growth rate. The growth rate of the wild type is not affected by the same treatment. The results are interpreted in terms of a red light absorbing intermediate of the blue light photoreceptor of Phycomyces. The intermediate should be short-lived in the wild type and should accumulate in madC mutants.     

Negative phototropism in the piloboloid mutants of Phycomyces blakesleeanus Bgff.

The sporangiophore (spph) of a piloboloid mutant, genotype pil, of Phycomyces ceases elongation and expands radially in the growth zone shortly after reaching the developmental stage IV b. The pil spph is always negatively phototropic to unilateral visible light when its diameter exceeds 210 m. Photoinduction of spph initiation, light-growth response, threshold of light energy fluence rate for the negative phototropism, avoidance and gravitropism in the pil mutant are all normal. In liquid paraffin, the pil spph shows negative phototropism as does the wild-type spph. Genetic analyses indicate that the negative phototropism of the pil mutant is governed by the phenotypic characteristics of pil but not by specific gene(s) responsible for negative phototropism. These facts imply that the reverse phototropism of the pil mutant results from a loss of the convergent lens effect of the cell because of the increase in cell diameter.Abbreviations spph(s) sporangiophore(s) - wt(s) wild type(s)     

Phycomyces: a new gene for a flavoprotein with covalently linked cofactor

Summary We have identified a new locus in Phycomyces blakesleeanus that codes for a flavoprotein designated previously as FP₂. According to results from a sexual cross, this locus, flp, maps near the gene madC, a marker for abnormal phototropic sensitivity. The recombination frequency between flp and madC is about 10%. Further, the flp marker is unlinked with the carotene locus carA. Because the flavoprotein FP₂ is absent in certain photoreceptor mutants (Pollock et al. 1985 a), it had been proposed as a candidate for the blue-light photoreceptor. We have found, however, that some strains lacking this protein retain normal phototropism. Therefore, the flavoprotein FP₂ is probably not involved in photoreception.     

A genetic map of Phycomyces blakesleeanus

Summary Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.     

Detoxification of N-(phosphonoacetyl)-L-aspartate by carrot cells in suspension culture

Phototropic reversal of Phycomyces sporangiophores can be elicited by a change to darkness during steady-state phototropism. The reversal lasts 25–30 min under these conditions. Control experiments show that the reversal is not caused by gravitropism. Tropic reversal is also elicited by the removal of a barrier during an avoidance response, showing that the reversal occurs at the output of the sensory transduction chain.     

Amylin evokes protein p20 phosphorylation and insulin resistance in rat skeletal muscle extensor digitorum longus

In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive labeling. The molecular basis of the amylin action was further examined using proteomic analysis. In particular, proteins of interest were characterized using an integrated microcharacterization procedure that involved in-gel trypsin digestion, organic solvent extraction, high performance liquid chromatography separation, microsequencing and microsequence analysis. We found that amylin significantly decreased the insulin-stimulated glucose incorporation into glycogen (p < 0.01) and produced a protein spot of approximately 20 ku in size. This amylin responsive protein (hereby designated as amylin responsive protein 1, APR1) was identified to be protein p20. Moreover, ARP1 spots on gels were found to consistently produce a corresponding radioactive spot on X-ray films in ³²Pi but not in ³⁵S-methionine labeling experiments. In conclusion, our results showed that in vitro amylin concomitantly evoked the production of ARP1 and caused insulin resistance in EDL muscle. It is suggested that protein p20 may be involved in amylin signal transduction and the appearance of ARP1 may be a step in a molecular pathway leading to the development of insulin resistance. ARP1 might therefore be a useful molecular marker for amylin action, insulin resistance and Type 2 diabetes.     

Analysis of microsomal flavoproteins from Phycomyces sporangiophores: Candidates for the blue-light photoreceptor

To help identify components of the blue-light photoreceptor system for phototropism in Phycomyces blakesleeanus Bgff., proteins from a microsomal fraction obtained from synchronous sporangiophores were studied. By two-dimensional gel electrophoresis, two proteins (FP₁, FP₂) with covalently bound flavins were found. FP₁ has a molecular weight of 71 000 and an isoelectric point of 6.6; FP₂ has a molecular weight of 59 000 and an isoelectric point of 6.5. These flavoproteins were purified by column chromatography and gel filtration while assaying for flavins by fluorescence. The relative concentrations of FP₁ and FP₂ were affected by light applied during growth. These flavoproteins are likely components of the blue-light photoreceptor complex mediating phototropism in Phycomyces.Abbreviations 10 k pellet 10 000-g pellet - 100 k pellet 100 000-g pellet - FP₁, FP₂ proteins with covalently bound flavins having molecular weights of 71 000 and 59 000 and isoelectric points of 6.6 and 6.5, respectively