Learning Journal 5

Your final project and your classmate’s final project

CJ and I will be reviewing each other’s portfolios, and I have posted the first part of my LJ5 on his blog.

• What is the most valuable thing that you learned from doing your Final Project (mini-research proposal)?

The most valuable lesson I learned is that you need to choose our research topic with care. The potential impact and the relevance is crucial for the success of the project. However interesting a topic might seem, a research project has no future if there is no potential for application or contribution to the field. I was surprised how complicated, time-consuming and expensive transgenic mouse assays are – if you are applying for funding for such a project, you need good reasons to explain the importance of the research.

• Look at what your classmate has posted under their Final Project section. What do you think your classmate may have learned, that you have not (and/or what do you think you have learned, and they may not have)?

CJ seems to have gained a lot from working with a completely new method – the UAS Gal4 system. I have expanded and developed my knowledge on knockdown assays in mice, but choosing an entirely new method would have been educating in a different way.

Learning Journal 4

1.     A technique

What is a technique that you learned about (or, that you learned more about) in BIOL463? Name it and briefly describe it.

RNA-TRAP is a method to investigate is an RNA of interest is in close proximity to a certain protein or a stretch of DNA, either in the process of transcription, translation or associated in other ways.

You add to your sample an RNA specific probe fused with an epitope for an antibody. The corresponding antibody is fused with HRP (horse radish peroxidase). When added, the antibody recognizes the epitope (hapten) and recruits HRP to the sites of the RNA.

By adding biotin linked with tyramide, HRP reacts with the tyramide to attach biotin to the adjacent proteins. This way, biotin is only added to the proteins in close association with the RNA. Using avidin (which has a high affinity for biotin) the biotin-linked proteins are pulled down. The proteins or the DNA associated with them can be sequenced or investigated in other ways.

2.     Anything difficult?

If someone were to learn how this technique works, what do you think would be the most difficult or confusing part, and why? (If you think nothing would be confusing, why is that?)

The difficult part lies in wrapping your head around the many components of the method. Realizing the purpose of all the elements can take a while, especially the many components that are required to recruit HRP to the RNA (probe, epitope, antibody).

3.     A question to test one’s understanding

If you were to test whether someone truly understands how the technique above works, what question would you ask them, and how would you interpret their answer (i.e., what answer would you consider evidence that they understand?)

I would ask them to explain the function of HRP in the method.

The most essential part is the understanding that by recruiting HRP to the RNA you can attach biotin to adjacent proteins and identify them (or the associated DNA).

Learning Journal 3:

One thing that stood out for you on the midterm or during your midterm preparation

This week you will write (or may already have written) your first midterm for the course. What is one thing that stood out to you either during your preparation for the midterm, or on/during the midterm itself? Is this something that had stood out to you before, or is it something new?

Briefly describe it and explain what made it stand out for you.

So far, genomic imprinting has been the most exciting topic for me in this course. I am familiar with the concept from a previous genetics course, but the mechanisms of regulation and the organization of the imprinted loci are completely new.

The most outstanding concept I encountered, is the regulating activity of transcription itself – unrelated to the transcript.

Transcription of the Airn locus effectively silences the overlapping Igf2r gene in cis, independently from the lncRNA transcript. This is a new dimension to the transcriptional process, which before (in my mind) only had the purpose of creating functional transcripts.

The regulating actions of long non-coding RNAs in relation to imprinting are also new to me, but it somehow seems less outstanding: It is similar to a very familiar concept of small non-coding RNAs and their many functions in the cell. I can’t think of such an analog to gene regulation by transcription of another gene, which is why I choose to put exactly this in my learning journal.

Learning Journal 2

1.     One new (or ‘improved’) concept

Now that we have looked a variety of experiments and techniques (e.g. honeybee papers, techniques presentations), some epigenetics mechanisms, the effects and characteristics of boundaries/insulators, etc., identify and briefly describe one concept that is either new for you, or that you understand better or in a new way.

In our class on Wednesday, we briefly discussed the concept of non-canonical histones, which is completely new to me. The presence of other variants of the histones adds a new dimension to my knowledge of histones and their role in expression of adjacent genes. This can be perceived as “improving” my knowledge on the concept of histones and chromatin structure, or as a “new” concept of variant histone proteins. Either way, it is quite interesting!

2.     Thinking about your new or ‘improved’ concept

• How did you identify your new or ‘improved’ concept? How did you decide that it is a concept (and not, for example, a fact or a skill or a technique)?

Looking through the lecture slides, I came across a note I had written about this concept. Initially, it seemed like a piece of factual knowledge (“histones come in several different variants”), but after some consideration, I decided it was rather a concept of gene regulation. It complicates and refines the pattern of chromatin changes and modifications affecting gene expression patterns in a more abstract way than factual knowledge would be able to.

• What made you realize/decide that you understand it better or in a new way?

Given the fact that there are multiple copies of histone genes in our genome, I find it curious that the idea had not occured to me previously – why wouldn’t the cells have exploited this opportunity to specify chromatin structure and gene regulation?

I find that learning something that subsequently seems obvious is a good indicator that the information fits in with/fills a gap in your previous knowledge.

• Do you think having a good understanding of this concept is important and/or useful? Why/how?

Knowledge of this concept is essential for gene expression studies involving chromatin. Only focusing on the canonical histones would be neglecting an entire area of chromatin-mediated gene regulation and could only reveal parts of the overall picture.

Learning Journal 1

1.     Factual knowledge

Briefly describe one new piece of factual knowledge that you acquired or developed since the start of the course.

I have learned the meaning of the term ‘specification’  in relation to cell differentiation and fate.

2.     Making connections (conceptual knowledge!)

How does this new piece of knowledge fit into what you already knew? What other facts is it connected to, and how? Does it fit into any concepts? How?

The two terms ‘specification’ and ‘determination’ are easily confused – at least for me! Both terms are connected to the potency and potential fate of a cell, but in two different ways.

As far as I understood, ‘specification’ describes whether a cell has committed to a certain line of development. If you extract the cell from its natural environment, it is still able to follow the line of development it had started without external signals. It is not necessarily determined.

Determination, on the other hand, refers to the state of the cell at a given time. The potency of the cell depends on the gene expression patterns (i.e. on epigenetics, chromatin structure etc.) that may or may not be reversible. A determined cell has undergone irreversible epigenetic changes.

These two terms complement each other well and fit into several concepts of developmental biology – for example, the distinction between regulative and mosaic development (which happens to be another piece of factual knowledge I have acquired) is largely based on the differences in specification and determination of the cells.

3.     Other comments (optional)

As I was working on this learning journal, describing the differences of the terms, I started feeling unsure that what I had understood was correct. I looked at the specification/determination slide and read a bit about the definition on Wikipedia – and realized that what I had written so far was completely untrue. So the way I learned the meaning of ‘specification’, was by reflecting on how I learned the meaning of term in the first place. Very metacognitive indeed.