Neurospora Lab

Introduction

• The circadian system exhibited by the ascomycete fungus Neurospora crassa lends itself well for use in the classroom for laboratory experiments aimed at introducing students to the basic tenets of chronobiology. As an organism, Neurospora displays many suitable attributes: it is non-pathogenic, grows on a simple defined medium, and exhibits a clearly defined clock output when growing on solid media in a petri dish. Stocks can be frozen at -20C (typical home freezer temperature) from year to year, and then quickly rejuvenated. Growth, transfer, and manipulation of the cultures requires only standard microbiological techniques: a clean surface, sterile growth media, inoculating loops, and a gas or alcohol flame.

• The circadian clock of Neurospora regulates the position of a developmental switch that serves to determine the mode of growth of cultures at the growing front of a colony or culture. That is, in the sense that development can be described as the controlled activation and deactivation of genes and gene products over time often accompanied by resultant morphological changes, Neurospora can be said to undergo a daily cycle of differentiation with the clock controlling the developmental switch. Clock assays are typically carried out in "race tubes" (hollow glass tubes about 40 cm long and 16 mm in diameter, bent up at both ends in order to hold an agar growth medium) although l50X15 mm petri dishes can also been used. A schematic view of a race tube is shown.

  

• Cultures of Neurospora will grow across an agar surface at constant rate (about 3 to 4 cm per day) reflecting the strain, temperature and nutritional richness of the medium. Following inoculation and growth for a day in constant light, the position of the growth front is marked and the culture transferred to constant darkness (LD transfer); the position of the growing front can be marked at 24 hour intervals thereafter in the presence of red light. Since Neurospora has a blue-light photoreceptor the clock in Neurospora "sees" white or blue light but does not perceive red light. During this subsequent period of vegetative growth, the biological clock of Neurospora regulates the timing of a physiological switch that ultimately controls the pathway of mycelial development in the region of the growing front. The LD transfer sets the clock running from circadian time (CT) 12 and sets the developmental switch such that mycelia, as they are laid down, are determined not to differentiate. Sometime later, at a time corresponding to late subjective night, the switch is thrown the other way so that mycelia as they are laid down are determined to differentiate. Then for several hours, the mycelia as they are laid down are endowed with the capacity to elaborate aerial hyphae which eventually (during subsequent days, long after the growing front has moved on) differentiate to produce asexual spores called conidia. As linear growth proceeds further down the race tube, after a time this developmental switch is reversed and the mycelia that are laid down no longer have the capacity to differentiate. The growing front thus leaves behind a band of differentiating hyphae morphologically and biochemically distinct from the surface hyphae on either side. This cycle recurs approximately every 22 hours (the duration of one circadian cycle), and once each region is laid down the hyphae are developmentally set. Thus, following a week of growth in constant darkness, an agar surface is covered by fluffy yellow-orange conidiating bands alternating with undifferentiated surface growth. Since growth rate is more-or-less constant for any strain over the course of the circadian cycle, distance grown (as determined by the daily growth marks) = time elapsed since the LD transfer. The assay of the clock in Neurospora is in this sense "selfautomating", since the period length and phase of the rhythm are simply read from this pattern of growth.

• This conidial banding pattern can be seen in wild type Neurospora, although it is often masked due to the fact that the elevated CO2 levels in race tubes or closed petri dishes tend to suppress conidiation. Fortunately, a mutation in the band gene was identified some years ago that results in the alleviation of this CO2 masking effect. For this reason, all commonly used lab stocks carry the bd mutation.

• An additional feature of the Neurospora system, of course, is the availability of various clock mutations. In the assay system described here, the results of the mutations appear as changes in the periodicity of the conidial bands; growth rates here are neither genetically regulated nor controlled by the clock and can vary from tube to tube depending on generally uncontrolled factors such as depth of the growth medium. Simply stated, long period mutants (with period lengths longer than a day) form conidial bands less frequently than the 24 growth marks appear, whereas short period mutants do so more frequently. Temperature compensation of the clock can be demonstrated by growing cultures at different temperatures between 20C and 30C, and another interesting facet of the long period mutations at thefrq locus is that they display partial loss in this temperature compensation feature, thus providing something interesting for students to "discover".

Set-up

• Neurospora cultures are easily grown and kept on the bench top at room temperature in disposable glass test tubes containing standard growth media (so called "slants"; see below for recipes). Transfer of cultures between slants, or from slants to race tubes or petri dishes is effected by using a sterile inoculating loop and carrying a few mycelia or some of the fluffy yellow spores from one culture to the place of inoculation. This inevitably results in some spores breaking free from the inoculum and floating off in the air. While there is no inherent safety issue with regards to the wind-borne dispersal of clouds of fungal spores as crowds of students attempt sterile transfers, many instructors prefer to limit this potential source of contamination. This is easily achieved by using genetically engineered strains in which spores do not break free from the mycelial surface (See Sargent, Neurospora Newsletter 32:12-13, 1985, for a brief discussion of Neurospora strains in the classroom .) Commonly used strains in this context carry the csp-l,csp-2 (conidial separation), or eas (easily wettable) mutations. Thus, a good beginning stock would be csp-l;bd or csp-2;bd or eas;bd.

• A beginning experiment for an initial lab experience. Plan to use petri dishes which are cheap, disposable, and readily available. A good growth medium that will support rhythmicity of cultures contains Vogel's salts (for basic salts, nitrogen, and vitamins) with 0.5% maltose as a carbon source, and solidified with 1.5% or 2% agar. Autoclave or otherwise heat sterilize this, and pour 50 mls to 60 mls into 150 mm X 15 mm petri dishes. As the medium cools, water will condense on the tops; this should be discarded when the agar is solid. Let the plates air dry upside down with the tops on for a day or so. Inoculate the plate on the side or in the center, and keep it in the light for a day. Growth will start and continue at a rate of a few centimeters per day. Now turn the plate upside down and use a magic marker to mark the position of the growing front across the plate; then transfer the plates from light to dark. Use a light-tight cupboard in a room that can be completely darkened. This will set the clock to subjective dusk (CT 12). During the next 8 or so hours, conidiation will be reduced, and then later, as the subjective time of the culture approaches late night, it will increase again. The next day, at the same time of day cultures were initially placed in the dark, darken room completely, turn on a lamp with a red light bulb (a good hardware store should cary them) and mark the position of the growth front on each culture. Repeat this every subsequent day at the same time of day until the cultures grow to the ends of the petri dish. When the cultures reach the edge of the plate, take them out of the dark and mark the positions of the centers of the conidial bands with a dot. Plot the positions of the growth fronts in mm versus day number; since growth rate is constant, the slope of this line or a quick calculation gives a figure for "hours/mm of growth". Now plot the positions of the conidial band centers, or simply tabulate them as distance in mm from the point of inoculation and use the Hrs/mm growth conversion factor to calculate the numbers of hrs between subsequent bands of conidiation. This is the period length of the clock.

Additional Studies

• For additional studies, all of the standard characteristics of rhythms can be demonstrated on these cultures. They can be reset by brief (2-5 minute) pulses of light to give a standard type O (strong) Phase response curve. The period length will remain fairly close to 22 hrs at all temperatures between 20C and 30C, thus demonstrating temperature compensation, but can be reset by brief steps (an hour or so) into higher or lower temperatures. Strains bearing various mutations in the clock are also available to demonstrate the genetic basis of period length. Order strain bd,frqZ for a 29 hr period length at 25C and bd,frq2 for a 19 hr. period length strain. These strains can also be crossed to demonstrate the genetic basis of the period length phenotype.

Acknowledgments

• Thanks to Stu Brody for suggestions on the petri dish cultures. This work was supported by grants from the National Science Foundation (MCB 9307299 to JJL), the Air Force Of fice of Scientific Research (to JJL), the National Institute of Mental Health (MH44651 to JCD), and the National Institute of General Medical Sciences (GM34985 to JCD).

Media:

• Vogel's salts - 50X (Vogel, HJ. 1956. Microbial Genetics Bull 13: 42-43; see also Davis and deSerres, Methods in Enzymology 27A: 79-143, 1970) In 750 ml dH20 dissolve successively. Make sure compound is dissolved completely before adding the next ingredient.
  • Na3 citrate . 5.5 H20 (2 H20) 150 g (125 g in 775 ml dH20) KH2P04 (anhydrous) 250 g NH4N03 (anhydrous) 100 g MgS04 . 7 H20 10 g (4.88 g MgS04) CaCl2 . 2 H20 (O H20) 5 g (3.8 g) Trace element solution 5.0 ml Biotin solution (0.1 mg / ml) 2.5 ml
  • Total volume: 1 liter

• Vigorous stirring helps dissolve ingredients in a reasonable amount of time (either large stir bar or large glass rod)

• pH to 5.8 (no adjustment should be necessary)

• Add ~2 ml chloroform as a preservative - store 50X strength stock at room temp.

• Autoclave single strength after adding carbon source (0.5% maltose, 2% sucrose or 1% sucrose and 1% glycerol). Glycerol alone not utilized as a carbon source in minimal medium.

Trace Element Solution for Medium N

• citric acid . 1 H20 5.0 g

• ZnS04 . 7 H20 5.0 g

• Fe(NH4)2 (S04) . 6 H20 1.0 g

• CuS04 . 5 H20 0.25 g

• MnS04 . H20 0.05 g

• H3B03 - anhydrous 0.05 g

• Na2MoO4 . 2 H20 0.05 g
  • Total volume is 100 ml

  • Add 1 ml chloroform as preservative. Store at room temp.

Genetic stocks

• Genetic stocks are available for a minimal fee, and free advice on the care and feeding of Neurospora is always available, from the FGSC (Fungal Genetics Stock Center ), a truly superior operation run by friendly people who actually care about their work.

• Fungal Genetics Stock Center
  University of Kansas Medical Center
  Kansas City, KS 66103
  FAX (913) 588-7044
  Phone (913) 588-7295
  The Director is Prof. Jack Kinsey, and the chief of stocks is Craig Wilson.

Further Reading

• Dunlap, Jay C. 1990. Closely Watched Clocks: Molecular Analysis of Circadian Rhythms in Neurospora and Drosophila. Trends in Genetics 6, 159-165 (cover article).

• Dunlap, Jay C. and Jennifer, J. Loros. 1990. Genetics and Molecular Genetics of the Circadian Biological Clock in Neurospora, Seminars in Developmental Biology 1, 221-232.

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