Circadian Clocks, Clock Genes, and Clock-Controlled Genes
Notes from Prof. Jay Dunlap, Biochemistry (2/96)
Introduction
- Circadian clocks
- overview and perspective
- definition
- Perspective
- used for lots of things; anatomical locus may reflect use
- behavioral rhythms have a focus in the nervous system
- caveat is that rhythmicity per se does not require neuronal complexity
- Comparative anatomy of circadian rhythms
- Unicells and fungi
- Primates - SCN (suprachiasmatic nucleus) - shown by lesioning
and transplantation; presumptive neuronal connection to rest of
organism
- lizards and birds - pineal gland - shown by lesioning, transplantation,
and in vitro culture; largely but perhaps not exclusively humoral connection
(via melatonin) to rest of organism
- insects - brain (for behavioral rhythms), neuronal or humoral
connection, depending on organism
- Drosophila (fruit fly) - brain, probably humoral connection - based on mosaic
analysis and transplantation
Genetics of clocks in general
- Why use genetics?
- Neurospora and Drosophila in brief overview
Neurospora
- Biology of the Neurospora clock
- Clock-Controlled Genes
- Isolation via subtractive hybridization
- ccg-1 and ccg-2 are transcriptionally regulated
- ccg-2 is a fungal hydrophobin
- Clock Genes and clock-affecting genes
General Information
- the frq gene
- how cloned and by whom
- the transcript and its regulation
- ca. 4500 nucleotides, no introns; long 5'UTR
- transcript level oscillates
- arrhythmic but high in null strains
- the protein and its regulation
- 989 amino acids (108,000 daltons); molecular hallmarks TG/SG repeat,
PEST
- probably contained within the nucleus
- basis of mutations
- protein level oscillates
- proving how the clock works
- how light resets the clock
Drosophila per and Per
- Biology of per
- Controls
- eclosion
- locomoter activity
- courtship song - genetic and behavioral basis
- Anatomical loci
- circadian clock in brain
- courtship song in thorax (by mosaic analysis)
- Temporal and anatomical limits of expression - both by in situ
hybridization and by beta-galactosidase fusions
- egg
- embryo (salivary gland? midline central nervous system)
- larval stages
- pupa (prothoracic gland, corpora allata, optic lobes)
- adult (antenna, proboscis, eyes, optic lobes, central brain cells,
thoracic ganglion, gut, Malphghian tubules, ovarian follicle cells
- extensive mosaic analysis by Ewer and Hall show that expression in any of a number of places
in the brain is sufficient to rescue rhythmicity; apparently not
just neuronal expression needed
- Genetics and Molecular Biology of per
- how cloned and by whom
- the transcript and its regulation
- 4500 nucleotides, 7 introns; nontranslated first exon
- possibility of complex splicing
- transcript level oscillates, requires 5' noncoding region of mRNA
- arrhythmic in null strains
- the protein and its regulation
- 1218 amino acids (molecular weight = 127,000 daltons); molecular
hallmarks include TG repeat
- largely but not completely contained within the nucleus; also
glial cells
- present in both neuronal and glial cells; neuronal expression
may not be essential for rhythmicity, although it may be essential
for robust rhythmicity
- per0 are all truncated proteins, perl and pers loss of function mutations; clearly interesting implications
for the latter
- includes conserved and nonconserved regions
- protein level oscillates in the eye; requires N-terminal end of
protein; is inherently capable of rhythmicity (i.e. constitutive
mRNA expression yields rhythmic protein)
- arrhythmic but high in null strains
- NB: cycling is inherent to protein. In the absence of the protein, the 5'
part of the gene will drive cycling of a beta-galactosidase reporter
at the mRNA level but not at the protein level
- similarity at the sequence level to sim and ARNT (aryl hydrocarbon receptor nuclear translocator protein),
and ARH in a domain of unknown function characterized by 51 aa
repeats separated by about 100 aa. Sim, ARNT and ARH, in addition,
share a basic helix-loop-helix domain typically associated with
transcription factors
- Timeless
- who identified it
- who cloned it and how they did it
- characteristics of the regulation of the gene and protein are
very similar to those of per
- TIM appears to be central to light resetting; flies are reset
in manner similar to but different from fungi
Models
- based on
- (1) evidence for negative feedback
- (2) clock gene cycling which phase lags per cycling by about 4-6 hours
- necessary modifications?
- frq is a morning specific gene and per is an evening gene
- more than one oscillator?
- Phylogenetic conservation?