Monthly Archives: April 2015

Why Assignments?

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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?

Final Learning Journal

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LJ Week 13

Three main things in BIOL 463

  1. Genetics is complicated, and we are still trying to piece it together.

Although I had some exposure to the world of genetics during my undergraduate degree, this class is the first that really tried to explore the huge variation in genetics and developmental pathways. In 1st and 2nd year, genetics is always taught in a Mendelian-manner which it gives us the impression that systems like Hox or Xist/Tsix are somehow ‘special’ and not usually the norm. However, it is clear after this term that ‘complicated’ is much more common than ‘simple’ when it comes to developmental genetics! Another thing that I realized this year was how little we truly understand about genetics so far. Reading papers and looking at them critically in class has changed my perspective of our progress in genetics. That’s not to say we haven’t come a long way (because we definitely have!), but rather that there is still much to be discovered.

  1. Look at the evidence and consider what it means—don’t be afraid to be wrong!

This course has done a wonderful job of teaching us how to look critically at papers. I feel like previous courses tended to concentrate on ‘factual’ information much more than ‘skills’, and I really appreciate the effort to incorporate the latter into the curriculum. The discussions in class were great for practising analytical thinking because it forced us to listen to other people’s points of view. It allowed us to continually improve our theories or hypothesis by listening to our classmates and I thought that was really neat. Finally, the class taught us that no answer is ‘wrong’—that as long as your model fits the data, there is no reason why it couldn’t be completely valid.

  1. Our bodies use all sorts of creative shortcuts and tricks to get done what needs to be done.

I feel like I’ve been exposed to so many neat systems this year! The use of progressive segmentation in hox genes, gene interaction in dorsal/ventral differentiation, and the strange Xist/Tsix system in X-inactivation are just a few examples of ways our bodies are able to tightly control gene regulation and development. Learning about the unique ways our bodies can do these things is a little inspiring—and I think it allows us to think outside the box when we are coming up with our own methods to our own problems.

Identify types of knowledge

  1. I feel like this is a cross between ‘Factual’ and ‘Conceptual’. We have learned that gene regulation during development is very complicated, and we have done so by exploring different systems by which our bodies regulate different functions. Thus, while it requires us to ‘conceptualize’ all the systems working in unity to create a single organism, we need the individual ‘facts’ about each system in order to create a complete image in our mind.
  2. This is a ‘Skills/Procedural’ type of knowledge. We have learned how to discuss, critique, and improve our hypothesis.
  3. I think this kind of knowledge is conceptual. In this course we have gained an overall understanding of the ways our bodies regulate growth and development. Although it is likely we will not remember the names and order of the genes involved in these processes, we will remember the general mechanisms that take place. Thus, we understand the concept of gene regulation and how different structures might be regulated differently.

What makes “things” stand out for you

  1. Everyday coming out of class, the thought “Wow, that was complicated” ran through my mind. So obviously, it is one of the main ‘messages’ from this course that is likely going to stay with me. The awe that I have for genetics and regulation is increased greatly, and that concept has stood out for me because I know it will stay with me for a long time.
  2. The ability to make, discuss, and improve our mental models is a skill that stood out for me because of its application in research (and probably other areas of life too!). I think there is this general contempt held by students regarding coursework because the material they learn in class seldom seems applicable or useful after their final. However, this class makes it you feel like it was ‘worth taking’ because it doesn’t emphasize crystalline knowledge—and it is this focus on concepts and skills that make the course really stand out.
  3. I consider myself to be a pretty creative person, and one of the things I loved learning about in this course is the absolutely genius ways our bodies have come up with to grow and develop! One of the things that draw me to research is the perpetual search for ‘solutions’ to our ‘problems’ and I think it’s really neat to learn about some of the solutions genetics and evolution has come up with to solve their problems.

Relevance/use

  1. I think that understanding the complexity of genetics helps shed the ‘genetic essentialism’ view of humans. Even being aware of the complexities associated with genetics can help someone think critically about the articles they read on Facebook, or see on the news, or hear from a friend.
  2. The skill to interpret, create, and improve models based on data are ones that will be really useful in my research career. Of course, these concepts can also be extended into everyday life as well. Critical thinking and the ability to listen to other people’s ideas is something that will improve our thought process in both research and life.
  3. I think being aware of the different ways our bodies have found to solve developmental hurdles will help me find solutions to my own problems in the future. There are many examples of new inventions or ideas being based on phenomena found in nature, so it is easy to see how being exposed to the processes in our bodies may help us find solutions to other problems as well.

Three main things in your undergraduate education

  1. “You can make it. Every previous time you thought you were going to fail, or die, or not make that darn deadline—you have. So stop worrying about whether you will fail and start thinking about how great it will be once you succeed” I am a worrier. I sometimes think I actually might like it, since I always seem to put myself in these situations. But I’ve realized that some things are not worth worrying about, because they cause me (and the people I love) unnecessary stress. My boyfriend was watching me freak out for a test one day when he told me this—and it dawned on me that he was absolutely right. I can make it. I mean, I’ve made it so far so who’s to say I won’t make it this time?
  2. “Hear what other people have to say, but listen to yourself.” Sometimes the most unexpected people can give you the most profound advice, and you should definitely learn from the people who have been there and done that—that being said, I think I’ve learned it’s really important to understand yourself. Sometimes it’s really hard to separate what other people expect of you from what you truly want to do, but I think it’s important to know that distinction. Everyone will have a different opinion on what you should do, where you should go, and how you should get there. Some of their advice will likely be helpful and some of it not so much. But ultimately you have to trust yourself. You know yourself better than anyone, and you should stand up for who YOU are.
  3. “Do it.” Take every opportunity you can. Don’t say no to something new. Seize the day. Take a chance on a whim. That course that sounds cool but is rated really difficult? –Take it. Offered a strange project opportunity that is different from your current field of study? –Why not? Life (and undergrad!) is short. It’s easy to regret not doing something, but very rare to regret finishing it.

Techniques Speed Dating

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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.

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Melissa is for “Honey Bee” :)

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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

 

  1. 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.

 

  1. 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

 

  1. 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

 

  1. 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

 

  1. 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

 

  1. 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.

 

  1. 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

 

 

  1. ***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