Modifying the Circadian Rhythm of Neurospora crassa Through the Administration of Environmental Chemicals

Francis A. Fahning
Department of Biology, Rutgers University, Camden NJ 08102


The  circadian  clock  is  a  necessary  component  for  the  regulation of daily cellular activities. Previous studies have  shown  that  pharmaceutical  chemicals  can  alter  the  period of the circadian rhythm of certain organisms. This study  was  constructed  to  determine  if  the  circadian  rhythm of Neurospora crassa, a filamentous fungus and a successful  eukaryotic  model  organism,  could  be  altered  through the use of chemicals that are easily accessible. The successful outcome could open avenues of developing drugs  that  would  modify  the  circadian  rhythms  of  eukaryotic organisms including humans.


The  circadian  rhythm  is  an  entrainable  cyclic  biological  process with a period of about 24 hours. Another characteristic  of  the  circadian  rhythm  is  temperature  compensation, meaning that the circadian rhythm will not change  when  an  organism  is  exposed  to  an  environment  of the physiological range of temperatures. Neurospora crassa  is  an  excellent  organism  in  characterizing  molecular mechanisms of the circadian rhythm (Baker 2012).  N.  crassa  is  classified  as  a  filamentous  fungus  which grows in a web like formation. Its usual habitat is in the  sub-­‐tropical  region  and  found  on  dead  or  decaying  plant material often after a forest fire. N. crassa is an ideal candidate  for  research  as  its  haploid  life  cycle  allows  for  simple tracking when used for genetic modification.

Circadian  rhythms  are  generated  by  a  circadian  oscillator, which involves transcription and translation of clock genes in a cyclic process. This process is organized of  coupled  negative  and  positive  feedback  loops.  Specific  to N. crassa is the frequency gene (frq) and white collar 1 (wc1) and white collar 2 (wc2) genes associated with the white  collar  complex  (WCC).  Within  N. crassa’s  circadian  oscillator, frq influences the period of the rhythm (Baker 2012). The gene expression of frq is regulated by wc1 and wc2. WCC binds to the frq promoter and up-­‐regulates the expression of frq in the early morning, a positive feedback loop. The protein FRQ begins and forms homodimmers to enter  the  cell  nucleus  to  repress  frq  transcription  by  inactivating WCC, a negative feedback loop. WCC is then removed  from  the  nucleus  and  initiates  the  negative  feedback loop beginning in mid-­‐day to late-­‐day (Baker 2012). Following the reduction of WCC, frq levels go down and the synthesis of FRQ decreases.

Recent  study  showed  that  there  is  a  metabolic  oscillator whose function is not dependent on transcription  or  translation  (O’Neill  2011).  Another  important finding of the same study was that pharmaceutical  chemicals  would  influence  the  metabolic  oscillator of O. tauri (a single cell organism) the same way as they would on that of human cells.
In  this  study  we  will  test  if  the  environmental  chemicals influence the N. crassa clock gene frq. We hypothesized  that  N.  crassa’s  circadian  rhythm  would  remain in a cyclic pattern with the chemicals having no affect. The significance of this experiment will ascertain if chemicals  can  influence  the  circadian  rhythm  and  how  great of an effect the chemicals can produce. The chemicals used are common to everyday people and may produce  results  beneficial  for  our  knowledge  as  eukaryotic circadian oscillators operate in comparable fashions.

Materials & Methods

The  experimental  design  of  testing  N. crassa was  similar  to the study performed on O. tauri (O’Neill 2011). This unique  experiment  consisted  of  testing  a  strain  of  N.  crassa’s frq gene as it is the clock gene that influences the period length of N. crassa’s circadian rhythm. The frq gene was  composed  with  a  suspension  of  high  glucose  media  and a luciferase bioluminescence reporter along with the administration  of  a  drug/chemical.  The  strain  was  designated N. crassa frqlv2 luciferase. The luciferase reporter  adheres  to  the  mRNA  transcriptional components of the frq gene and allows the ability to track protein synthesis.

Six  conditions  were  used  in  the  form  of  common  chemicals containing stimulant properties. The purpose was to prove the hypothesis wrong and I believed the best choice was to illustrate this through the use of well known stimulants.  The  six  chemicals/conditions  used  were  pseudoephedrine, phenylephrine, two cigarette brands containing nicotine and various other chemicals, caffeine, and coffee.
Pseudoephedrine,  primarily  a  nasal  decongestant,  is  also classified as a stimulant and ergogenic aid decreasing the effects of fatigue and has been widely used by athletes (Gillies  1996;  Hodges  2006).  When  compared  to  pseudoephedrine, phenylephrine (a substitute nasal decongestant)  is  to  have  a  reduced  effect  on  the  adrenergic receptors of the sympathetic nervous system in  mammals  and  both  chemicals  may  show  a  differing  result when administered to N. crassa. Nicotine is mainly absorbed  through  tobacco  products  and  has  stimulant  properties.

The  two  tobacco  products  used  in  this  experiment  were Pall Mall cigarettes (containing additives) and Natural  American  Spirit  Organic  cigarettes  (organically  grown and without additives). Several other chemicals known  as  additives  are  contained  within  a  cigarette.  These chemicals have been approved by the FDA as food additives and may attribute to a variety of unpredictable effects. Caffeine is a well-­‐known stimulant used to combat fatigue and can be found in beverages and pill form. The methods  of  caffeine  administration  for  the  experiment  consisted of caffeine in tablet form and caffeine through coffee.  Caffeine  and  coffee  were  compared  as  coffee  contains chemicals such as monoamine oxidase inhibitors, which possess anti-­‐depressant qualities, and β-­‐carbolines norharman  and  harman,  which  are  alkaloids  which  may  produce psychoactive effects (Herraiz 2006).

The experimental set-­‐up was as follows:

Pseudoephedrine: One 30mg tablet crushed with a mortar and  pestle  into  a  powder. Pseudoephedrine  powder  mixed into approximately 50ml of water at 30°C. Chemical powder sat for thirty minutes to dissolve.

Phenylephrine:  One  10mg  tablet  crushed  with  a  mortar  and pestle into a powder. Pseudoephedrine powder mixed  into  approximately  50ml  of  water  at  30°C.  Chemical powder sat for thirty minutes to dissolve.

Caffeine:  One  200mg  tablet  crushed  with  a  mortar  and  pestle into a powder. Pseudoephedrine powder mixed into  approximately  50ml  of  water  at  30°C.  Chemical  powder sat for thirty minutes to dissolve.

Coffee:  Approx.  25ml  of  Starbuck’s  Pike’s  Place  Medium  Roast coffee was obtained directly.

Pall Mall cigarette: The tobacco contents of one cigarette were removed and placed in a “cheese-­‐cloth” type fabric. Approximately  50ml  of  water  was  raised  to  boiling  temperature and the tobacco was added to create a solution.

Natural  American  Spirit  Organic  cigarette:  The  tobacco  contents of one cigarette were removed and placed in a “cheese-­‐cloth”  type  fabric.  Approximately  50ml  of  water  was raised to boiling temperature and the tobacco was added to create a solution.

200µl of 2% high glucose media was prepared for six replicates  of  each  condition.  Each  replicate  (pseudoephedrine, phenylephrine, Pall Mall cigarettes, Natural American Spirit cigarettes, caffeine, & coffee) was titrated  with  200µl  of  media  and  200µl  of  the  chemical  giving the 1:1 (full strength) ratio. A high glucose media was produced to provide nutrients for the N. crassa frqlv2 luciferase  strain.  The  mRNA  transcriptional  bioluminescent reporter luciferin was also added along with the strain to track frq gene transcription levels. A 12 replicate  N.  crassa  frqlv2  luciferase  strain  with  no  chemicals added was used as the control.

The  plate  was  then  placed  in  a  luminometer  in  constant darkness for 96 hours to record gene transcription  levels  through  the  bioluminescence  of  luciferase reporter at 30 minute intervals. The luminometer measures in relative light units (RLU) as the system  does  not  translate  measurements  into  units  of  photons. The RLU’s signifies the level of frq mRNA transcription that occurs. The assay was performed for 96 hours.  The  luciferase  activity  reflects  the  frq  mRNA  level  in the cell. There were six replicates for each strain and 12 replicates  of  the  control  without  any  chemicals. 


First,  we  wanted  to  test  if  the  selected  environmental  chemicals affect the levels of the key clock gene frq. Using luciferase-­‐bearing N. crassa, we measured the changes of frq levels (see Materials and Methods). The average levels of  frq-­‐luciferase  levels  are  summarized  in  Figure  1.  The  RLU averages are reported as: pseudoephedrine-­‐1077.30 RLU,  phenylephrine-­‐3770.03  RLU,  Pall  Mall  cigarette-­‐7289.96 RLU, Natural American Spirit cigarette-­‐7700.56 RLU,  caffeine-­‐380.71  RLU,  coffee-­‐4692.20  RLU,  and  the  control-­‐2686.29 RLU.

All  chemical  groups  show  statistically  significant  differences from the control group (t-­‐test p<0.001). To further verify the findings an ANOVA test was applied to the  results  to  determine  if  a  significant  result  occurred  between all groups. All groups had statistically significant difference  at  a  value  of  p<0.0001  with  the  exception  of  both cigarette groups (Pall Mall and Natural American Spirit).  When  each  cigarette  group  was  compared  to  any  other group there appeared to be a significant difference, however,  there  was  no  difference  in  frq-­‐luciferase  levels  between Pall Mall cigarette and the Natural American Spirit  cigarette  (p=0.39).  The  comparison  was  made  between cigarettes to find if a difference in ingredients would significantly alter the transcription level of the frq gene.  Two  other  comparisons  were  made  between  pseudoephedrine and phenylephrine, and caffeine tablet and  coffee.  Results  showed  pseudoephedrine  group  compared to phenylephrine group and caffeine tablet group  compared  to  coffee  group  all  proved  to  be  statistically significant at values p<0.01.

The  phenylephrine  group  showed  to  maintain  a  circadian rhythm with a similar period of the control group.  Pseudoephedrine  showed  a  significantly  low  rhythm with a diminished period (Fig S1). The two cigarette  groups  were  compared  along  with  the  control  (Fig S2). The Pall Mall cigarette group and the Natural American  Spirit  cigarette  group  follow  a  close  circadian  rhythm for the first day up until day two. Shortly after the second day the Natural American Spirit group displays an arrhythmic period and in approximately the middle of day two  the  Pall  Mall  cigarette  group  also  begins  an  arrhythmic period. Near the start of the third day both cigarette groups display an inexplicable rhythm. Normally a  period  occurs  once  a  day,  however,  for  both  cigarette  groups the period occurs approximately three times on day  four.  The  caffeine  group  and  coffee  group  were  compared along with the control group to reveal contrasting  differences  in  N.  crassa’s  circadian  rhythm.  The period displayed by the coffee group matches closely to the control group maintaining a steady rhythmicity (Fig S3).  Surprisingly  the  caffeine  group  lacked  a  noticeable  rhythm.

Figure 1. A  graph  showing  the  average  transcription  levels of frq gene as reported by the luciferase reporter. Six  replicates  from  each  chemical/condition  were  averaged in as well as the 12 replicates of the controls strain. The error bar represents standard error.


This study demonstrated that the common environmental chemicals  used  can  influence  the  level  and  the  circadian  rhythm of the frq gene, a key molecular component, in N. crassa’s circadian rhythm. The chemicals used in the study are  known  to  have  stimulant  effects  on  mammalian  physiology. The frq gene being part of the negative feedback loop of N. crassa’s circadian oscillator, normally shows increasing transcription levels from mid-­‐day to late day  (Baker  2012).  Pseudoephedrine  and  caffeine  were able to suppress the frq gene essentially decreasing the negative feedback loop producing no discernible rhythm. Phenylephrine  and  coffee  produced  a  similar  rhythmic  pattern to that of the control which could mean the circadian oscillator was able to function correctly without inhibiting  either  the  positive  or  negative  feedback  loop.  Pseudoephedrine and phenylephrine being entirely different  chemicals  could  explain  the  drastic  change  of  rhythm they produced. Coffee and caffeine share some of the  same  properties  as  caffeine  is  a  chemical  within  coffee, but coffee contains other chemicals such as monoamine  oxidase  inhibitors,  and  β-­‐carbolines  norharman and harman which may provide an explanation  as  to  the  continued  rhythmicity  of  the  frq  gene. Further tests may be applied with monomine oxidase  and  β-­‐carbolines  to  determine  how  frq  transcription would react in regards to coffee. The cigarette  brands  Pall  Mall  and  Natural  American  Spirit  showed very similar effects. With Pall Mall containing additives and Natural American Spirit absent of additives and  organically  grown,  both  resulted  with  no  significant  difference in RLUs or circadian rhythm. However, it is a curious anomaly as both cigarettes showed arrhythmicity towards  the  second  half  of  the  96  hour  experiment.  This  severe interference recorded may be due to the stimulant drug,  nicotine,  or  a  variety  of  other  chemicals  present  within cigarettes. As no additives were declared in the Natural  American  Spirit  brand,  and  both  showed  similar  results, it can be surmised that the results were due to nicotine.  Supplementary  testing  of  nicotine  alone  would  need to be applied to compare if a variance in frq transcription  or  a  change  in  the  period  of  the  circadian  rhythm occurs to discern if nicotine is an influencing factor.    Further  investigation  would  be  required  to  determine how these chemicals interact with other components  of  N.  crassa’s  circadian  oscillator.  Future  results of studies of this nature may give more insight on how  chemicals,  which  are  commonly  used  in  our  daily  lives, are affecting the circadian rhythms of other eukaryotic organisms including humans.


I’d  like  to  thank  Dr.  Kwangwon  Lee  for  his  patience  and  guidance every time I came to his office with a dumbfounded look of confusion on my face. I’d also like to thank my wife, Sarah, for her encouragement and support through  the  late  nights/early  mornings  of  study  and  research. Lastly, I’d like to thank Mike O’Brien for helping me  format  graphs  in  excel.  The  study  was  performed  as  part of the course requirement for General Microbiology Laboratory at Rutgers University – Camden.


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