Nstojan1 Week 12

1. Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. You can use the glossary in any molecular biology, cell biology, or genetics text book as a source for definitions, or you can use one of many available online biological dictionaries (links below). Cite your sources for the definitions by providing the proper citation (for a book) or the URL to the page with the definition for online sources. Each definition must have it's own citation, to a book or URL. Make an in text citation of the (name, year) format next to the definition, and then list the full citation in the References section of your journal page.
   1. Blotting: The transfer of protein, rna, dna molecules from a relatively thick acrylamide or agrose gel or to a paper like membrane (usually nylon or agarose gel) by capillarity or an electric field, preserving the spatial arrangment. Once on the membrane, the molecules are immobilised, typically by baking or by ultra violet irradiation and can then be detected at high sensitivity by hybridisation (in the case of dna & rna) or antibody labelling (in the case of protein). Rna blots are called northern blots, dna blots, southern, protein blots, western (Biology Online, 2022).
   2. Filamentous growth: The process in which a multicellular organism, a unicellular organism or a group of unicellular organisms grow in a threadlike, filamentous shape (AmiGO 2)
   3. In vitro: (Of a biological process) made to occur outside the living organism, i.e. in an artificial environment such as within a glassware, a test tube, etc. (Biology Online, 2021)
   4. Respiratory electron transport chain: A process in which a series of electron carriers operate together to transfer electrons from donors such as NADH and FADH2 to any of several different terminal electron acceptors to generate a transmembrane electrochemical gradient. (AmiGO 2)
   5. Reading frame: One of the three possible ways of reading a nucleotide sequence. As the genetic code is read in nonoverlapping triplets (codons) there are three possible ways of translating a sequence of nucleotides into a protein, each with a different starting point. For example: given the nucleotide sequence: AGCAGCAGC, the three reading frames are: AGC AGC AGC, GCA GCA, CAG CAG (Biology Online, 2022).

1. Answer the following questions about your article. Your answers need to be in YOUR OWN WORDS, not copied straight from the article. It is not acceptable to copy another student's answers either. Even if you work together to understand the article, your individual entries need to be in your own words.
   1. What is the main result presented in this paper?
   • The main result presented in the paper is that they were able to identify the DNA sequences bound by transcriptional regulators under different growth conditions. They were also able to classify the regulatory binding behaviors into condition-invariant, condition-enabled, condition-expanded, and condition-altered. They discussed the different mechanisms for the different binding behaviors such as nuclear exclusion and increased protein levels and why the regulatory elements were organized the way they were in yeast promoters.
   1. What is the importance or significance of this work? This work is important because it discusses the advancement in understanding the regulatory mechanisms that controls gene expression in yeast. Under the different growth conditions, they were able to identify the specific DNA sequences that were bound by transcriptional regulators which provided a map of the transcriptional regulatory code.
   2. What were the limitations in previous studies that led them to perform this work? Previously, they weren't;t able to map the transcriptional regulatory code because there was limited understanding if the specific DNA sequenced that were bound by transcriptional regulators. They also didn't understand how the different growth conditions affected transcriptional regulators which was important in order to understand the regulatory mechanism. There was also little understanding about regulatory elements and how they were used by regulators in different environments in yeast promoters.
   3. How did they treat the yeast cells (what experiment were they doing?) The yeast cells were treated in a couple of ways: with the Myc-epitope coding which allowed for all of the 203 regulators to be tagged for further analysis. Crosslinkng and Immunoprecipitation, cross linking stabilized interactions between the target DNA sequence and transcriptional regulators, using immunoprecipitation, they were able to isolate specific DNA fragments that were tagged with the myc-tagged regulators. Then the ligation-medicated PCR was used to augment the DNA for further analysis. Using microarray hybridization, they were able to detect the DNA sequence that was bound by transcriptional regulators in the yeast.
   4. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid? A haploid Saccharomyces cerevisiae W303 strain.
   5. What media did they grow them in? What temperature? What type of incubator? For how long? PCR was used to grow the microarrays. The cells were also grown on YPD media. The temperature is not specified in the paper except for the elevated temperature environmental condition which was 30 degrees and then 37 degrees celsius. The time was different depending on the growth conditions and media. The times ranged from 20 minutes to 14 hours.
   6. What controls did they use? Their control was unenriched DNA samples according to the supplementary methods.
   7. How many replicates did they perform per treatment or timepoint? The supplementary methods states that the program was ran 50 times on random sequences on sets of different probes.
   8. What method did they use to prepare the RNA, label it and hybridize it to the microarray? To prepare, they crosslinked the DNA which was then immunoprecipitated. They then reversed the crosslink in order to be able to separate the DNA from protein. Unenriched samples were then amplified and labelled with a ligation-mediated PCR. They hybridized it to the microarray by spotted PCR products.
   9. What mathematical/statistical method did they use to analyze the data? Probability scores were used to determine the significance of the binding interactions.
   10. Are the data publicly available for download? From which web site? The raw data can't be downloaded.
   11. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
   • Figure 1: Identifies and maps which specific DNA sequences are bound by yeast transcriptional regulators. The ChIP experiment indicated which DNA sequence is bound to transcriptional factors in yeast cells. The Phylogenetic conservation data shows different sequences in different species of yeast that are important for the binding. The figure also lists different motifs that are binding sites for the transcriptional factors.
   • Figure 2: Is a more detailed transcriptional regulatory map. It represents the interactions between different DNA sequences and transcriptional factors they bind to. It also shows the chromosome and how they are distributed among the different sections in yeast.

     Figure 3: It shows the arrangement of regulatory sequences in yeast DNA to control gene expression.

   • Figure 4: shows the different patterns of DNA binding behavior of transcription regulators in the different environmental conditions.
   • Figure 5: The main finding is that the binding or regulators to promoters is specific and selective.
   • Figure 6: This figure shows the way a large number of motifs are filtered to identify more precise motifs. The key finding was that 65 motifs were assigned to an equal number of regulators. This was reduced from 283 motifs and 82 regulators.
   • Figure 7: The main findings discovered that in Cin5, the binding site was specific for the newly found DNA motif that was compared to a previous site.
   • Figure 8: In a more nutrient rich environment some regulators bind mire to promoter regions compared to when there were little to no amino acids present. Regulatory activity is condition invariant.
   • Figure 9:Gcn4 tightly binds to DNA under normal conditions, however, it becomes more relaxed under amino acid starvation. It also indicates that there might be a different gene regulation in response to the stress from the environment.
   1. • What do the X and Y axes represent (if applicable)? For figure 5, the x-axis is the regulators in the order of the number the promoter regions bind to. The y-axis is the promoter region that's bound to each regulator. In figure 9, the x-axis is the different regulators and the y-axis is the amount of promoter regions that are bind each of these regulators under the 2 conditions.
   2. What future directions should the authors take? Perhaps they could expand the genomic analyses and use different types of yeast species to see the evolution of regulatory and transcriptional elements. It could help discover other regulatory motifs across different species of yeast.
   3. Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any major flaws to the paper? I think that they supported their conclusions well with the data, however a major flaw in the paper is that the data was not explained well. It would be almost impossible for someone to replicate the experiment without looking at the supplementary methods and for a paper like this, the materials and methods section was too short. While the order of the tables does make sense and you can draw connections, the fact that they combined several data types, they could have done a better job at explaining. At first, the paper was extremely hard to understand unless you are in the biology field. The figures were also hard to understand and they never really state their most important findings, there isn't a result or conclusion, after every "topic" they throw in the main findings which makes it confusing for the reader.

Bibliography Questions

1. Harbison, C. T., Gordon, D. B., Lee, T. I., Rinaldi, N. J., Macisaac, K. D., Danford, T. W., Hannett, N. M., Tagne, J. B., Reynolds, D. B., Yoo, J., Jennings, E. G., Zeitlinger, J., Pokholok, D. K., Kellis, M., Rolfe, P. A., Takusagawa, K. T., Lander, E. S., Gifford, D. K., Fraenkel, E., & Young, R. A. (2004). Transcriptional regulatory code of a eukaryotic genome. Nature, 431(7004), 99–104. https://doi.org/10.1038/nature02800
2. [1]
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006441/
4. [2]
5. [3]
6. The copyright is owned by the Author because it is an Author Manuscript
7. Once I open the full text, I do see "Public Access".
   • The article is open access.
   • Accessing it from NIH was free, however to access it from "nature" it says that it is a subscription content and must be accessed via the institution
8. The journal Nature is available for print, since it is available to subscribe to print.
9. The publisher of the Journal is Nature Portfolio, which is part of Springer Nature, they are for profit. They are not a member of OAPA.
10. Since 1989
11. Yes, the articles in this journal are peer-reviewed.
12. [4]
13. 64.8 was the 2 year impact factor and 60.9 was the 5 year impact factor (2022).
14. The article was submitted on March 11 2004
15. The article was accepted on July 1st 2004
16. No/unknown, all it says is that it was published in final edited form 2004, Sept 2.
17. The article was published on September 2 2004
18. 7 months
19. Whitehead Institute of Biomedical Research, Massachusetts Institute of Technology, MIT Computer Science and Artificial Intelligence Laboratory
20. One of the authors, Christopher T Harbison, had published a paper in 2002 on Transcriptional Regulatory Networks in Saccharomyces cerevisiae. He also published a paper on Genome-wide map of nucleosome acetylation and methylation in yeast in 2005. Another author, D Benjamin Gordon, also published a paper relating to transcription factors in 2004, as well as An improved map of conserved regulatory sites for Saccharomyces cerevisiae in 2006.
21. Yes, “Some authors have filed a patent application covering aspects of this work and are pursuing commercialization.”
22. There isn't data associated with the dataset.
23. This article has cites 30 articles, and has been cited by 1671 articles.