Techniques speed dating:

I spent a lot of time making the poster! I like the way it turned out overall, and I thought it was really fun to do. Also, I finally learned the difference between qPCR, RT-PCR, real time PCR, etc.

 

SRS and BWS assignment:

I felt like Kimmy, Enzo and I worked really hard on this assignment. It was kind of confusing and we really gave it our best. Here’s the pushing ourselves beyond our comfort zones!

 

Assignment 1:

I took quite a while to come up with these– and did quite a bit of reading on each of them as well to make sure they were all ‘supported’. So I’m pretty happy with this assignment.

 

Melissa is for Honey Bee:

A really neat lecture in general! I liked the ‘pick-your-own-adventure’ feel to the class, and I thought the content was really interesting. Also, my name is relevant to the assignment– so what’s not to love?

TECHNIQUES SPEED-DATING: Quantitative Polymerase Chain Reaction

Names and contributions of group members:

Kenrick Ocampo-Tan (Editing, answering questions)

Natasha Tripp (added a few bullet points, edited references)

Lorenzo Luis Casal (editing, answering questions)

Melissa Chen (Editing, condensing information, poster design)

Kimmy Wong (Organized documents, added several bullet points, editing info)

Technique chosen: Quantitative PCR and its derivatives

 

What does this technique ‘do’?

• Quantitative polymerase chain reaction estimates the starting copy number of a DNA/mRNA segment
  • Can be estimated using the final product concentration after ‘X’ cycles (end-point qPCR) or by detecting the amount of products generated after each cycle (real-time qPCR)¹

What applications are this technique employed for?

• Estimating DNA copy number or detecting DNA from a sample²
• Gene expression analysis by quantifying mRNA (RT-qPCR)^2,4

 

What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?

• How does the copy number of a certain gene compare with others? (qPCR)
• What housekeeping genes are active during specific time in development of fetal animals? (RT-qPCR)
• What genes are used in development but rarely expressed in adulthood? (RT-qPCR)
• Is there differential expression of genes under different stress conditions? (RT-qPCR)

What critical reagents are required to use this technique?

• Both qPCR and RT-qPCR require standard PCR reagents (Forward and reverse primers, dNTPs, thermophilic polymerase, Mg2+, appropriate buffers)
  • qPCR also requires DNA template and either a dye or probe that fluoresces when hydrolyzed or hybridized⁴
  • RT-qPCR requires reverse-transcriptase (and other standard RT-PCR reagents) in addition to all of the above
• real-time PCR requires a PCR machine capable of detecting the amount of product after each cycle

What critical information is required to be able to employ this technique?

• Sequence data to be able to create primers
• Proper annealing temperature for reaction specificity

References:

 

1. Heid, Christian A., Junco Stevens, Kenneth J. Livak, and P. Mickey Williams. “Real time quantitative PCR.” Genome research10 (1996): 986-994. Web. 01 Feb 2015.
2. Karlen, Yann, Alan McNair, Sébastien Perseguers, Christian Mazza, and Nicolas Mermod. “Statistical significance of quantitative PCR.” BMC bioinformatics1 (2007): 131. Web. 01 Feb 2015.
3. Lederman, Lynne. “QPCR.” BioTechniques4 (2009): 817-19. Web. 01 Feb 2015.
4. VanGuilder, Heather, Kent Vrana, and Willard Freeman. “Twenty-five Years Of Quantitative PCR For Gene Expression Analysis.” Biotechniques 5 (2008): 619-26. Web. 01 Feb 2015.

SRS_BWS

Epigenetics and Development

 

Our Goals:

• To build our own definition of “epigenetics”;
• To classify the major mechanisms of chromatin remodeling;
• To investigate how external environment/behaviour can trigger a completely different developmental fate
• To propose, and interpret some experiments that allow us to shed light on this phenomenon (in a specific case)

 

 

1. What could be the underlying mechanism driving the different developmental trajectory of the organisms studied in today’s class?

DIET plays a huge role in determining whether a bee becomes a queen or a worker. It may act via signalling pathways, hormones, or DNA methylation. These things may then change gene expression that will be characteristic of either a Queen or a worker

Other options could be some kind of juvenile hormones—perhaps queen bees are pre-designated and produce hormones that prevent others from being queen and promote their own queen-growth

 

2. a) With one or two partners, take two minutes to come up with a definition of “epigenetics”:

A non-sequence related difference in gene expression

1. b) After listening to other classmates’ ideas, provide a more complete (if necessary) definition of “epigenetics”:

• important to realize that epigenetics does not necessarily have to do with GENES. Can be something in the cytoplasm; methylation, histone, etc etc
• everyone agreed epigenetic is non-sequence or base-pair related… but it doesn’t even have to do with DNA t all.
• “Inheritance of a phenotype that is not explained via DNA differences”à for example, tetraformena (a protist) can inherit the direction of mouth bristles BUT it has nothing to do with genetics! It’s the way they divide the cell during mitosis.

 

3. a) What are different mechanisms that can affect developmental trajectory, and that could be affected/directed by an “outside factor”?

• coiling/supercoiling
• enzymes involved with histone modifications
• factors involved in mitosis
• acetylase/methylation
• different environments might sensitize or desensitize these pathways

 

1. b) What are the mechanisms that can affect chromatin structure?

• histone variants
• ATP-dependent chromatin structure remodeling
• Post translational modifications
• DNA methylation

 

4. What would you predict about gene expression patterns in the two distinct developmental trajectories if epigenetics is driving the phenotype?

• sex developmentà workers have reproduction shut off
• growth hormones
• telemerases
• serotonin/reward genes associated with working habits

 

5. Researchers (Grozinger et al., 2007) actually checked… what do you notice about the gene expression patterns in individuals following each of the two developmental trajectories?

• Worker bees have upregulation of foraging-related genes and down regulation of reproductive/longevity genes
• Queen bees have the exact opposite: lots of upregulated reproductive and longevity genes but down regulation of foraging ones

 

6. What kind of protein/factor could be a key component of the epigenetic control of developmental trajectories? How would you test your hypothesis?

• Could be CPG islands
• Could be methylationà do ChiP of different histones (H3K4,9,27)
• Could be DNA acetylationà inject larvae with DNA acetylase inhibitors
• Could be other proteinsà test whether they are necessary by injecting larvae with iRNA

 

7. What did Kucharski and colleagues find, and what does their experiment suggest?

• Found that by inhibiting Dnmt via RNAi, you can induce Queen-like traits
• This suggests that workers had methylated DNA while queen has unmethylated DNA
• Suggests that maybe worker phenotype is “default”? Because it takes methylation of worker DNA to make a Queen phenotype.

 

8. a) What component of the food in question is most likely to affect gene regulation?

Lipidà specifically, Royal Jelly Acid

 

1. How does the food in question activate a transcriptionally silenced gene?

It affects the transcription of the Fas gene. The Fas gene is a positive regulator of cell division, and it is up-regulated in Royal Jelly-treated larvae

 

 

9. ***optional*** Using these pieces of data that we just discussed, construct a model of how consumption of the food in question leads to each of the developmental trajectories.

Day 2: Wednesday 1 April 2015

Review:

Queen bee receives royal jelly

Royal jelly acid is a part of royal jelly

There is differential gene expression in Queen and worker bees

If you treat bee larvae with Royal Jelly acid, you can induce queen symptoms

If you knock down Dnmt using iRNA, you can cause an increase of development in queen-like phenotypes (reproductive things).

 

Using these pieces of data that we just discussed, you can try to construct a model of how royal jelly consumption lead to Queen bee development.

• Royal jelly acid is absorbed into the larvae through ingestion
• Due to polarity, it diffuses into cellsà likely gets digested a little
• Interacts with proteins responsible for activating Dnmt proteins
  • Perhaps it interacts with receptors that activate a cascade of kinases that activate or deactivate Dnmt
• This in turn methylates DNA to shut down “Worker bee” genes
  • One of these genes might be an inhibitory lncRNA (like Xist)
  • Lacking lncRNA, queen-like traits will become activated
• Thus, the default is worker bee but the queen develop if Royal jelly blocks.

 

Other suggestions:

• jelly causes increase histone acetylation
• histone acetylation then causes queen bee genes to be more expressed

 

Are there other things in royal jelly that affect final phenotype?

• other than jelly acid, probably other nutrients
• nutrition and nutrient-rich diets can affect phenotypes later on; not necessarily bc of active component, but because of nutritional component.
• You could isolate royal jelly acid and feed bees nectar+acid; royal jelly – acid; assess phenotype