Experiments on Circadian Rhythms using the Easily Visualized Circadian Rhythm in Conidiation of Neurospora crassa

Jennifer J. Loros, Deborah Bell Pedersen* and Jay C. Dunlap
Department of Biochemistry
Dartmouth Medical School
Hanover, NH 03755-3844

Internet Jennifer.Loros@Dartmouth.EDU

* current address
Department of Biology
Texas A&M University
College Station, TX 77843-3258

Prof. Kathleen L. Triman (717) 291-3948
Department of Biology
Franklin and Marshall College
P.O. Box 3003
Lancaster, PA 17604-3003 USA

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 has 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 150mm test tubes 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 is marked at 24 hour intervals thereafter. 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 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 21.5 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, 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 "self-automating", 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 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 the frq locus is that they display partial loss in this temperature compensation feature, thus providing something interesting for students to "discover".
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 test tubes is effected by using a sterile inoculating look 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-1, csp-2 (conidial separation), or eas (easily wettable) mutations. Thus, a good beginning stock would be csp-1; bd or csp-2; bd or eas; bd.

A beginning experiment for an initial lab experience. Plan to use 150mm test tubes 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 sodium acetate (1.2%) as a carbon source, and solidified with 2% agar. Autoclave or otherwise heat sterilize this, and pour 8 mls into 150mm test tubes. Cap tube and cool in horizontal position. A quick shake of the tube will minimize condensation forming inside the tube, as the medium cools. Let the test tubes dry with caps on for a day or so. Inoculate the tube and keep it in the light for a day. Growth will start and continue at a rate of a few centimeters per day. Now use a magic marker to mark the position of the growing front across the tube; then transfer the tubes from light to dark. (Darkness is actually controlled red light. We use widely available fluorescent F40R red lights (40 Watt red fluorescent tubes))
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. Mark the position of the growth front on each culture the next day and on every subsequent at the same time of day. When the cultures reach the end of the tube take them out of the dark and mark the positions of the centers of the conidial bands. 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.

Experiments can be documented with software from the NIH and a scanner. A description of this approach is also available from the FGSC at www.fgsc.net/teaching/imgcirc.htm

For additional studies, all of the standard characteristics of rhythms can be demonstrated on these cultures. They can be reset by brief (2 minute) pulses of light to give a standard type 0 (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, and can be crossed to demonstrate the genetic basis of the period length phenotype.


. TThis work was supported by grants from the National Science Foundation (MCB 9307299 to JJL), the Air Force Office of Scientific Research (to JJL), the National Institute of Mental Health (MH44651 to JCD), the National Institute of General Medical Sciences (GM34985 to JCD), and a Visiting Scholar Award from Project Kaleidoscope, Washington, D.C. to KT.


Vogel's salts - 50X
In 750 ml. distilled water, dissolve successively with stirring at room temperature:
Na3 citrate, 5 1/2 H20                 150 grams
KH2PO4, anhydrous                      250 grams
NH4NO3, anhydrous                      100 grams
MgSO4, 7 H20                            10 grams
CaCl2, 2 H20                             5 grams

Add with stirring

Trace Element Solution (see below)       5 ml.
Biotin Solution (see below)              2.5 ml.

The resulting total volume is about 1.00 liter. Chloroform (2 ml.) is added as a preservative, and the 50 times strength medium obtained is stored at room temperature. For use, this medium is diluted 50 fold with distilled water. The resulting single-strength medium is designated N; it has a pH of about 5. 8. Medium N is supplemented with a suitable carbon source (0.5% maltose, 2% sucrose or 1% sucrose and 1% glycerol). Glycerol alone not utilized as a carbon source in minimal medium. The thus supplemented medium is sterilized by autoclaving.

The trace element solution (containing citric acid as a solubilizing agent) is made up as follows: In 95 ml. distilled water, dissolve successively with stirring at room temperature:

                  Citric acid, 1 H20         5. 00 grams
                  ZnSO4, 7 H20               5. 00 grams
                  Fe(NH4)2(SO4)2, 6 H20      1. 00 gram
                  CuSO4, 5 H20               0. 25 gram
                  MnSO4, 1 H20               0. 05 gram
                  H3BO3, anhydrous           0. 05 gram
                  Na2MoO4, 2 H20             0. 05 gram

Sodium acetate conidial banding medium
H2O 100 ml
Vogel's salts 2ml
sodium acetate(anhydrous) 1.2g
5% casamino acids 0.5 ml
agar 2 g

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 Missouri-Kansas City
5007 Rockhill Road
Kansas City, MO 64110
Phone (816) 235-6485
FAX (816) 235-6561


The Director is  Dr. Mike Plamann, and the curator of stocks is Dr. Kevin McCluskey. The FGSC has a web site with information about Neuropora and many other filamentous fungi, including recipes and culture techniques.
The FGSC's Web site can be found at http://www.fgsc.net

Undergraduate Teaching Lab Schedule
day one: Inoculation of race tubes.
day two: Transfer of race tubes from light to dark . Begin 24 hour clock
day three: first Mark of growth front under red safelight at 24 hours.
day four: second Mark of growth front under red safelight at 24 hours.
day five: third Mark of growth front under red safelight at 24 hours.
day six: fourth Mark of growth front under red safelight at 24 hours.
day seven: Video transfer and storage of race tube image to computer in Imaging Lab

Example of observations and calculations
Raw data
Distance from point of inoculation
day growth front conidial band
1 119 166
2 213 243
3 296 325
4 394 409

Regression Plot
y =mx + b
y value is distance; x value is day; slope m = y/x; b= y intercept
y = 90.8x + 28.5
Growth distance/24 hour day = 3.783 per hour
(distance between growth front marks: 94, 83, and 98 on day 3, 4, 5, respectively are unitless numbers generated by computer)

(Between Conidial bands)
*Calculated Period (hrs) Average Period (hrs)
77 20.3 21.39
82 21.67
84 22.2

* period is calculated by multiplying distance between conidial bands by the inverse of slope (distance per hour determined from plot of distance between growth fronts versus 24 hour days).
Mating type strain number (FGSC) frq allele published
circadian period
circadian period
A 1858 frq + 21.5 hrs. avg=21.4 hrs.

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