Bio154JM08/Toolbox/Lecture 14
This page is part of the BIO154/254 Experimental Toolbox!
Lecture 14 Model Systems
Drosophila eclosion
Eclosion is the emergence of fruit flies from their pupas. Even when pupas are moved into complete darkness, their emergence happens at a fairly consistent point in time, at 'dawn'. This indicates that there is an internal rhythm within the fruit fly that is not affected by the constant darkness.
Mouse locomotor activity
Mouse locomotor activity, specifically running wheel activity, is useful for identifying circadian rhythm defects. In Lecture 14, mouse running wheel activity was measured in constant dark (DD) for 60 days as an example of self-sustained circadian rhythm in the absence of environmental cues (Pittendrigh 1993). The heavy bars of the actogram represent wheel utilization, while the period between activity onsets is slightly less than 24 hours. Using a forward genetic screen, Takahashi and colleagues were able to identify a semi-dominant mutant Clock in a mouse with a period of 24 hours. The homozygous mutant had a longer initial rhythm of 27 hours and lost rhythmicity completely after shifting to DD.
Golden hamster
The golden hamster is one of the most frequently used laboratory animals in chronobiological studies mainly because of its very strong and predictable rhythms. A spontaneous mutation in a Golden hamster was found to result in a significant decrease in the period of its circadian rhythm. The mutation occurs at a single gene locus called tau. Whereas a normal wild type hamster has a 24 hour period, a hamster heterozygous for the tau mutation has a 22 hour period, and a hamster homozygous for the tau mutation has a 20 hour period. When the tau mutant was discovered in 1988 by Ralph and Menaker, it was the first single gene mutation that causes an effect on circadian rhythm periodicity to be described in a mammalian species. Recently it was found that tau is an allele of the gene casein kinase I epsilon in the golden hamster. The tau mutation provided the opportunity to further test the hypothesis that SCN is the circadian pacemaker. When SCNs were transplanted between wild type and tau mutant hamsters the period of the host's circadian rhythm was always the same as the period of the donor's transplanted SCN. Thus a tau mutant with a wild-type SCN graft showed a 24 hour rhythm while a wild type hamster with a mutant SCN graft showed a 20 hour rhythm. From these findings it was concluded that the SCN contains all the necessary information for the proper functioning of circadian rhythms.
Lecture 14 Techniques
Yeast two-hybrid
The yeast two-hybrid system is commonly used to study protein-protein interactions between two proteins. This system takes advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. The GAL4 protein is a transcriptional activator that is required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG for upstream activating sequence of GAL4); and a C-terminal domain containing acidic regions necessary to activate gene transcription. In other words, GAL4 protein consists of two domains: GAL4 DNA binding domain (GAL4-DBD) and GAL4 activating domain (GAL4-AD).
In the two-hybrid system, you create two hybrid proteins: protein X bound to GAL4-DBD (otherwise known as the “bait” fusion, containing the protein of interest) and protein Y bound to GAL4-AD (the “prey” fusion, containing the potential interacting partner of the protein of interest). These fusion proteins are created in plasmid vectors and transfected into a host yeast cell. If protein X interacts with protein Y, the binding of these two will reconstitute the proximity of the GAL4 domains, forming an intact and functional transcriptional activator. This newly formed transcriptional activator will bind to the UASG, which regulates a reporter gene, a gene whose protein product can be easily detected and measured (e.g., LacZ, luciferase, GFP). In this way, the amount of the reporter produced can be used as a measure of interaction between the protein of interest and its potential partner (Fields and Song, 1989).
In Lecture 14 on circadian rhythms, Professor Luo presented results that showed that mClock does not form dimers with itself, instead binding to a second bHLH-PAS domain protein, bMAL, which was identified using a yeast two-hybrid system (slide 20). Furthermore, a yeast two-hybrid assay was also used to determine that dClock binds to the Drosophila homolog of bMAL, Cycle.
ZT
ZT stands for Zeitberger time. The number represented after the ZT indicates the hours since dawn.
Serum shock
Serum shock is the introduction of large amounts of foreign serum into cells. Injecting fibroblasts with high levels of serum can cause a fluctuation in protein levels that follows a circadian-like rhythm that can persist for many days.
DNA microarray
DNA microarray is a method for profiling gene expression levels for thousands of genes simultaneously. A DNA chip is made by designing florescent probes that match parts of the sequence of known mRNA. After inserting each desired mRNA reporter into the organism, microscopic DNA spots are attached to the solid chip surface. Each spot fluoresces according to the level of reporter binding to mRNA. To compare expression levels between two conditions (e.g. comparing gene expression between clock mutant and wildtype) it is necessary to create two chips, one for each strain. Observing which reporters decrease in expression with the mutant DNA chip will reveal which genes are affected by the loss of Clock function. (see McDonald and Rosbash 2001).
From Lecture 15, Slides 37 and 38: Because biotech companies are capable of making DNA chips that contain the entire 13,500 genes in Drosophila, it is possible to do genome wide analyses to identify which genes undergo circadian cycle. Experimenters can cluster genes according to their phases (high day expression, high night expression, etc.), and identify which genes are direct or indirect targets of central clock regulators (such as the Clock or bMAL). Microarray analysis in flies has already revealed that circadian genes fall into several large groups of proteins, including detoxification enzymes, ligand binding/carrier, transporter, immune/host defense genes and neuropeptide modulaters.
Tissue graft
Tissue grafting involves surgically transplanting tissue from one organism to another, or from one part of an organism to another part. The tissue is transplanted without a blood supply and must thus obtain blood from the new vascular bed to survive. Grafting is often used in medical procedures to replace damaged tissue, ranging from skin to bone. Grafting is also used in biological research to see the effects of foreign tissue on the host organism. There are three specific categories of grafts, xenografs, allografts and isografts. A xenograft involves the transplanation of tissue from one species to another, an allograft involves two heterogenic members of the same species and an isograft involves two isogenic members of the same species (ie. twins or clones).
In slide 15 of lecture 14 Professor Luo mentions a study done by Ralph et al. in 1990 in which grafting of the suprachiasmatic nucleus (SCN) was used to see whether the genotype of the SCN alone was sufficient to produce rhythmicity in a golden hamster. This procedure was an allograft since two different genotypes of the animal were used. One was the wildtype (~24 hour rhythmicity) and one was a homozygous tau mutant which exhibited a shorter ~20 hour rhythmicity. When the SCN of the wildtype was ablated and replaced with a mutant SCN, the circadian rhythm changed to the mutant phenotype of 20 hours. When the SCN of the mutant was ablated and replaced with a wildtype SCN, the circadian rhythm changed to the wildtype phenotype of 24 hours. Thus the study showed that the genotype of the SCN determines the rhythmicity of the animal.
Note: Slide 15 mentions heterotopic grafting, which may be an error since heterotopic grafting involves transplanting tissue into a region of the organism in which it did not originally belong (eg. putting the ear of one organism on the foot of another). However, Ralph et al. 1990 placed the foreign SCN in the same position of the brain which originally held the host SCN before it was ablated. Thus this seems to be an example of an isotopic graft.
ENU
ENU is also known as N-ethyl-N-nitrosourea (chemical formula C3H6N3O2) and is a very potent and powerful mutagen (Russell et al., 1979). It is used in mutagenesis screens to identify mouse behavioral mutants using forward genetic approaches (mutagenizing genome first, then observing resulting phenotype). On a molecular level, the chemical works by transferring the ethyl group of ENU to nucleobases (usally thymine) in nuclei acids.
ENU is often used to study circadian behavior in mice. Male mice are treated with the highest dose of ENU that can be tolerated without causing infertility. The primary target of ENU in the germline of mice is the spermatogonia, which can be highly mutagenized and go on to produce mutant gametes (ENU induces about 1 mutation in every 700 gametes). Mutant mice with germ lines that display mutant circadian phenotypes are then bred and gene function is explored.
Phase response curve
Plotting phase response curve is a way to reveal the relationship between a circadian phase shifting drug or treatment, and its effect on circadian timing. A standard PRC describes changes of the phase of the circadian rhythm in response to a brief pulse of light. A variety of agents aside from light are also capable of phase shifting the mammalian clock in the SCN (e.g. 5-HT agonists, histamine, melatonin, glutamate, neuropeptide Y, vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP)). Testing how various treatments effect the circadian clock at different moments of the subjective day or night give insight into the nature of the clock mechanism. An example is the gated nature of the photic effect on circadian timing in all organisms studied (Lecture 14, Slide 25). During the subjective day, a pulse of light has no significant change to the phase. In the first half of the subjective night, a pulse of light results in a phase delay, or shift back to the beginning of the night. In the second half of the subjective night, a pulse of light results in a phase advance, or shift to the beginning of the morning.
