Differential regulation of the foraging gene associated with task behaviors inharvester ants By Jonathan Kahn, Andrew Hoadley, and Claire Spitzer
Summary This paper is a first time series analysis of foraging gene expression in harvester ants. It shows that the task-specific expression patterns of foraging are aligned with the circadian rhythm of the ants. This data underlines the importance that time-related expression studies are extremely important and thus can be useful in explaining mechanisms that influence behaviour; such as foraging.
Background The individual behavior of a harvester ant is one thatchanges over their lifetime; Transition from nest workers to foragers as they age. This social organization is highly implicated(?associated with?) by a genetic pathway that involves the cGMP-activated protein kinase gene, foraging.
cGMP-activated Protein Kinase Gene
This gene is associated with behaviour in the harvester ant (also many other species-used as an example) and is conserved amoung species although the expression patterns and functions of the gene vary across taxa.
Background Continued Many species such as the honeybee and the fruit fly provide examples as to how important the foraging gene is for behaviour and implications on the species structure; such as division of labour. The foraging gene becomes much more interesting to look at after the discovery of task-specific expression in alignment with the circadian clock(?).
Background Continued This experiment looked to compare the amino acid sequence of foraging across social insects and observed whether the differential regulation of this gene is directly associated with task-related behaviours.
Methods The major techniques utilized during this experiment include: Deep freeze storage of samples qPCR/RT-PCR DNA sequencing/analysis
Deep Freeze Storage Upon collection of individual ants, specimens were immersed in liquid nitrogen and stored at -70°C until dissection This step is essential for the accuracy of the study and preservation of target RNA Deep freeze storage of samples ensures: Desired expression of RNA given time of sampling Prevention of degradation of “foraging” RNA
qPCR Otherwise known as quantitative PCR (also abbreviated RT-PCR) This technique was utilized in order to quantify the expression of the 130bp foraging gene at given intervals for which mRNA was extracted What is normal PCR?
qPCR (Traditional PCR) Once mRNA is extracted from the specimen, designed primers and DNApolare added to the extraction to create cDNA (complimentary DNA) of the target gene With PCR (polymerase chain reaction), primers and a high-temperature optimum DNApol are added to the extraction above to create DNA This mixture is then heated and cooled repeatedly to amplify the DNA
qPCR (Traditional PCR)
qPCR (Traditional PCR) However, this technique usually takes 20-40 cycles and can be used as a semi-quantitative tool at best since attachment of primers to DNA is not consistent So What is the difference between PCR and qPCR?
qPCR With qPCR, a fluorescent probe is used As DNApol elongates the target gene during cycles, the probe fluoresces and this is detected by a machine After multiple cycles, the machine’s log of relative fluorescence detection can be analyzed to obtain an accurate idea of the mRNA that was originally present
qPCR Traditional PCR reveals results at the end which are unhelpful in wanting to quantify gene expression qPCR, however, provides real time results which may be traced backwards to yield the level of mRNA that was originally present, essential to the evaluation of forgaing expression at a given time for this experiment
DNA Sequencing/Analysis A machine reads the target DNA that was developed by using Sanger sequencing principles Using ddNTPs (A, T, G, and C nucleosides) which both fluoresce (by differing color) and terminate elongation, the machine can assemble the overlapping fragments of newly synthesized DNA according to fluorescence to reveal the sequence This is then further analyzed by inputting the sequence into a global database, found at www.nih.gov
Catalytic Domain Effector Terminal Domain Foraging Protein Sequence