Learning Journals are a tool commonly used in professional schools (medical school, nursing school, teacher college, etc.) and in the humanities both to help learners engage in metacognition and to help instructors evaluate students’ learning.

Think about the work you did for BIOL463 so far (in and out of class, formally and informally), then try to address each question to the best of your abilities. You can then copy and paste your answers in your wordpress blog. Please do not build a new page, but rather add this LJ above or below your LJ1 entry.

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.

One things that stood out for me in preparation for the midterm was the emphasis on answering exactly what the question is asking, and being concise in data interpretations. I’ve found that exam questions are not the type that prompt memorized anwers of facts or details. Instead, I have noticed that understanding figures and experimental data and being able to interpret this information is much more valuable. I’ve found that it’s very important to distinguish between what the data show (exact description of what you see), and what you can conclude from it (what the data prove). In addition, neither of these questions suggest providing a mechanism that COULD possibly describe the data. Its important to never jump to conclusions about mechanisms or cause/effect relationships in data interpretation exam questions. This is something that has been mentioned many times in previous classes, however, I feel as though the testing style for this class emphasizes it even more.

Learning Journals are a tool commonly used in professional schools (medical school, nursing school, teacher college, etc.) and in the humanities both to help learners engage in metacognition and to help instructors evaluate students’ learning.

Think about the work you did for BIOL463 so far (in and out of class, formally and informally), then try to address each question to the best of your abilities. You can then copy and paste your answers in your wordpress blog. Please do not build a new page, but rather add this LJ above or below your LJ1 entry.

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.

How insulators can regulate gene expression is a brand new concept for me. Before this course, I only knew of transcription factors, enhancers, or inhibitors regulating gene expression but never insulators. How insulators work is they can flank the coding region of a gene, or exist directly adjacent to a gene and block effects of any nearby enhancers on that gene. The result of having an activated insulator is decreased or completely silenced transcription of the gene. How insulators can be activated or deactivated is through their methylation or demethylation. The effect of methyl groups added to an insulator can differ case to case (sometimes activate and sometimes deactivate).

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)?

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

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

I identified insulators as a new concept because never before have I seen a molecule or component of the genome act to ‘block’ the effects of another transcription regulator. Normally I have seen mechanisms that either directly enhance or directly inhibit transcription. However, this is the first time I have seen interactions between components to produce a result (change in gene expression).

I realized I understood the concept of insulators when I was able to predict the expression of a gene in two different situations: one with an active insulator and one  with an inactive insulator, where both had enhancers for the gene nearby.

Having a good understanding of insulators is useful because it helps us understand another one of the many ways in which gene transcription is regulated.  When assessing data of gene expression levels under varying conditions, having insulators present in the expression mechanism could give rise to particular results. Knowledge of this concept can help us interpret experimental data with much more accuracy.

TECHNIQUES PRESENTATIONS

1.   Names and contributions of group members:

Emily Clarke

Elena Giuchici

Sina Sahebpour

2.   Technique chosen:

ChIP (Chromatin Immunoprecipitation)

3.   What general biological, chemical, and/or physical principles and concepts is this technique based on?

Protein-DNA Interactions

4.   What does this technique ‘do’?

• ChIP is a technique used to identify the sequences where certain proteins bind to and interact with specific DNA sequences in vivo.

5.   What applications is this technique employed for?

• ChIP can be used to determine the specific location of various histone modifications in the genome, therefore identifying the targets of histone modifiers.
• ChIP allows the researcher to determine whether specific proteins are associated with specific regions of the genome.
• Proteins that are not bound directly to DNA or that depend on other proteins for binding activity in vivo can be analysed with ChIP.
• ChIP can be used to analyse the binding of proteins such as transcription factors on promoters or other DNA binding sites.
• Virtually any protein that interacts with DNA can be identified through ChIP experiments.

6.   What questions relating to gene regulation and/or development can be addressed using this technique? Provide two examples (peer-reviewed papers) that use this technique.

One question that can be answered is whether or not a set of genes are regulated by a specific transcription factor. The paper called “ A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos” isolates gene areas associated with the AGL15 transcription factor to analyze and sequence the genes that bind and become regulated by it, through the use of ChIP technology.

Another question that can be investigated is an association of histones with genes. ChIP allows researchers to study how regulation of a specific gene works by looking at how tightly the histones are bound to the gene of interest. The paper called “Direct Examination of Histone Acetylation on Myc Target Genes Using Chromatin Immunoprecipitation” uses ChIP to isolate the DNA of interest and study the level of acetylation on histones, and therefore understand how regulation of the Myc genes works.

 

7.   What critical reagents are required to use this technique?

Protein of interest

Chromatin from nuclei of your organism’s cells

Formaldehyde to crosslink the protein to the DNA

Antibody specific to the protein of interest

Beads to bind antibody (usually Streptavidin magnetic beads or agarose beads)

PCR Kit

 

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

• The protein of interest that is believed to interact with a specific area of DNA
• An antibody specific to that protein

9.   At least two resources/information sources that you recommend for learning more about this technique (one or more may be developed by your group):

1. Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends in biochemical sciences, 25 (3): 99-104.
2. R&D SYSTEMS a biotechne bran. N.p., 11 Dec. 2002. Web. 3 Oct. 2016. <https://www.rndsystems.com/resources/protocols/chromatin-immunoprecipitation-chip-protocol>.

 

10.   List of references consulted:

References:

1. Chromatin Immunoprecipitation (ChIP) Protocol. (2002) [Online]. Available from: https://www.rndsystems.com/resources/protocols/chromatin-immunoprecipitation-chip-protocol [Accessed 10/02 2016].
2. Eberhardy, S., D’Cunha, C. and Farnham, P. (2000) Direct examination of histone acetylation on Myc target genes using chromatin immunoprecipitation. Journal of Biological Chemistry, 275 (43): 33798-33805.
3. Hawkins, R.D., Hon, G.C. and Ren, B. (2010) Next-generation genomics: an integrative approach. Nature Reviews Genetics, 11 (7): 476-486.
4. Kuo, M. and Allis, C. (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in chromatin environment. Methods-a Companion to Methods in Enzymology, 19 (3): 425-433.
5. Lee, T.I., Johnstone, S.E. and Young, R.A. (2006) Chromatin immunoprecipitation and microarray-based analysis of protein location. Nature Protocols, 1 (2): 729-748.
6. Mardis, E.R. (2008) The impact of next-generation sequencing technology on genetics. Trends in Genetics, 24 (3): 133-141.
7. Nelson, J.D., Denisenko, O. and Bomsztyk, K. (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nature Protocols, 1 (1): 179-185.
8. Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends in biochemical sciences, 25 (3): 99-104.
9. Park, P.J. (2009) ChIP-seq: advantages and challenges of a maturing technology. Nature Reviews Genetics, 10 (10): 669-680.
10. Wang, H., Tang, W., Zhu, C., et al. (2002) A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos. Plant Journal, 32 (5): 831-843.