Robert J. Lowry

Accumulation of microfilaments in a colonial mutant of Neurospora crassa

A morphological mutant of Neurospora crassa, snowflake, is shown to contain filaments which are about 70 A in diameter, and up to several microns long, and which usually bunch in groups of a few to several hundred. They may be found longitudinally or transversely arranged with respect to the long axis of the cell and, in many cases, they run up to the plasma membrane, but not through it. The filaments often are arranged in crystalline arrays but may also be found as separate filaments. Sometimes the filaments are closely appressed to nuclei and may be found inside them. It is likely that the filaments are not the result of the dissociation of microtubules and are most likely microfilaments like those found in other organisms. Their relationship to the origin of certain morphological mutants in Neurospora is discussed.

A model for rhythmic and temperature-independent growth in ‘clock’ mutants of neurospora

The Q10 for the frequency (number of bands per 24 hours) of the ‘clock’ mutant (strain CL11A) ofNeurospora crassa over the range 20–30° C is close to 1.0. By contrast, that for the double mutant, ‘wrist watch’ (strain CL12a), is closer to 2 over this temperature range. Strain CL12a differs from ‘clock’ in other ways as well, including 1) decreased rate of linear extension and band size, 2) greater sensitivity of growth rate to high temperatures and, 3) masking of rhythmic growth below 15° C. The response to temperature of several colonial mutants and standard (‘wild-type’) strains was studied and it is shown that some strains are temperature-independent yet arhythmic. A temperature-compensation model is presented to explain the response of ‘clock’ mutants to temperature and it is concluded that they demonstrate a non-circadian free-running endogenous rhythm.

Activation of Neurospora Ascospores by Organic Solvents and Furans

It has been shown previously (Emerson, 1948; Sussman, 1953) that dormant ascospores of Neurospora respond to very low concentrations of heterocyclic 5-membered ring compounds, including certain thiophene and furfural derivatives, and pyrrole. Although over 100 other substances were tried, only compounds with the above structure were active, with the exception of ethyl ether which was weakly effective in amounts 100-fold greater than were required when heterocyclics were used. Since that time, it was discovered that temperatures between 40 and 48? C, although insufficient to activate the ascospores, enhanced their sensitivity to chemical activators (Sussman, 1954a). Therefore, it seemed reasonable to expect that a more detailed investigation of the chemical specificity involved in the activation process might be possible by the use of this technique. Moreover, the observation that ethyl ether was also an activator suggested the possibility that other organic solvents might also serve in this way. That this surmise was correct is shown by the following experiments, which expand the list of chemical activators to include aliphatic alcohols, esters, and ketones, in addition to the furans used previously and in the present studies.

Lysozyme-Induced Sensitivity of Neurospora Ascospores to Furfural

The precise nature of the permeability barriers of plant cells has eluded analysis although their physiological effects have been exhaustively studied. Contributing most to the difficulties encountered in these studies has been the complexity of their chemical constituents and or? ganization. However, an excellent approach to this problem exists in the use of enzymes as specific reagents in the analysis of cell wall and membrane constituents. Apart from their specificity, these substances act under mild (physiological) conditions and are large enough in size so that it may be assumed that their effect would be localized on the outside of the cell. Weibull (1953) has shown that the ability of a bacterial cell to retain certain cellular constituents is greatly altered by the use of lysozyme. In addition, Tomcsik (1954) reported that prolongations of bacterial cross walls may extend into the capsule on the basis of enzymatic digestion of antibody-treated cells. With these facts in mind, studies were undertaken on the effect of enzymatic treatment upon the ascospores of Neurospora tetrasperma. These cells exist in at least two different physiological conditions, the durations of which are under the control of the investigator. Thus, in the dormant state, the cell metabolizes at a low rate, is impermeable to some poisons, and is resistant to drying and other physical treatments. Two means exist for breaking dormancy and these include heat-treatment (Goddard, 1935) and treatment with furfural and related heterocyclic compounds (Emerson, 1948; Sussman, 1953). Upon such activation a twenty to thirty-fold increase in metabolic rate ensues, accompanied by other changes in permeability and resistance to chemical and physical treatments. Using these facts as a basis, the effect of lysozyme and other enzymes upon the activation and germination of ascospores of N. tetrasperma was investigated. By this means it was hoped to learn

The Number and Morphology of Moss Chromosomes

The time that suitable cytological material is available can be extended by bringing mosses into the laboratory in late winter and keeping them in covered glass dishes placed in strong diffuse light. Best growth of the gametophyte occurs at 68-70° F. and at a relative humidity just high enough to prevent the leaves from curling. Fruiting plants can be maintained in the laboratory in the same way but at a slightly lower relative humidity. Tissue for the study of the gametophytic chromosome complement is obtained from the stem apex and embryonic leaves. Sporophytic mitoses are obtained from the meristematic tip of developing sporophytes. For the study of meiosis, the columella, to which the spore mother cells adhere, is removed from the capsule. The tissues are fixed in acetic alcohol for 1–3 hours and then stained and squashed. Ways in which moss tissue reacts differently to the conventional squash methods are discussed and special directions are given.

Physiology of the Cell Surface of Neurospora Ascospores. II. Interference with Dye Adsorption by Polymyxin

Abstract Polymyxin B prevents the germination of ascospores of Neurospora tetrasperma with an LD 50 of 3–4 p.p.m. The toxic effect of polymyxin is partially reversed by calcium and magnesium ions. The effect of polymyxin on the respiration of activated ascospores does not become apparent until after 2 hr. The effect upon respiration is reversed by calcium ions. Dormant and activated ascospores remove about the same amount of polymyxin from solution almost immediately upon exposure to the antibiotic. After 90 min. the apparent uptake of activated cells is markedly diminished while that of the dormant spores increases slightly. Polymyxin competes noncompetitively with methylene blue for sites on the surface of the cells when added simultaneously with the dye. However, if the antibiotic is added before methylene blue, dye uptake is almost entirely suppressed and an uncompetitive type of inhibition results. These data, in conjunction with those showing polymyxin uptake by cell-wall fragments, suggest that the antibiotic occupies sites on the surface of the ascospore and that these sites differ from those binding methylene blue.