Falghane Week 3

Purpose

• read and analyze a scientific article.
  • be knowledgeable about the effects of cold environments "cold-shock" in the transcription of prokaryotes genes.
• interpret data from figures correctly and be able to present the information interpreted.

Preperation For Journal Club 1

Biological Terms Defintion

1. Budding: The formation of an outgrowth from an organism, and is capable of developing into a new individual; gemmation (Customers, 2005).
2. Chaperones: A family of cellular proteins that mediate the correct assembly or disassembly of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures(Customers,2005).
3. Shine-Dalgarno: A short stretch of nucleotides on a prokaryotic mRNA molecule upstream of the translational start site, that serves to bind to ribosomal RNA and thereby bring the ribosome to the initiation codon on the mRNA (Customers, 2005).
4. Desaturase: Any of several enzymes that put double bonds into the hydrocarbon areas of fatty acids (Customers,2005).
5. Reciprocal: The quotient arising from dividing unity by any quantity; thus 1/4 is the reciprocal of 4; 1 (Customers, 2005).
6. Oxidative: Capable of oxidizing other substances (Customers,2008)
7. Repressed: The inhibition of a gene's expression, this is typically caused by the change in the activity of a regulatory protein (Customers, 2005).
8. Hyperosmotic: A condition in which the total amount of solutes (both permeable and impermeable) in a solution is greater than that of another solution (Customers, 2008).
9. Trehalose: A disaccharide made up of two α-glucose molecules, and serves as an energy source in certain fungi, bacteria, plants, and invertebrates (Customers, 2018).
10. Induction: The act or process of inducing or causing to occur, especially the production of a specific morphogenetic effect in the developing embryo through the influence of evocators or organizers or the production of anesthesia or unconsciousness by use of appropriate agents (Customers,2005)

Outline of the Article

Experiment Result and the Significance of the Work

• The main result presented in this paper is how the model yeast cell decrease in their transcription rate when they are in low-temperature environments. However, they still adapt to the environment by changing their gene expression. Different genes contribute to this process and influence it.
• The significance of this work is that it shows cells response to alterations in the environment, and the extent to which cold environment affect the yeast cell. The result from it could be applicable to other cells like human cells and bacterial cells which helps in understanding cellular diseases and infections more.
• The limitation in previous studies that led to this work is that most studies were exploring the effect of cells being in heated/ hot environment comparing to a normal temperature environment. While this study explores the effect of a cold environment in yeast cells.

Experiment Done On the Cell:

• Cultures were grown overnight in YPD medium at 30°C and then transferred to a 10°C environment and was incubated. Samples were taken before harvesting to ensure that each culture has the same physiological state. It was then harvested after it reached a final OD600 of 0.6-0.8.
• Hot Phenol method was used to isolate RNA.
• Reverse transcription was used for mRNA resulting in cDNA which was hybridized into DNA microarrays and the microarray slides were analyzed.
• Control Microarrays were grown at in 30°C cultures.
• The strains of the yeast cells that were used were BY4743 and BSY25 and they were diploid.
• Timepoints of 0,2, and 12 were replicated twice whereas timepoints of 10 min, 30min, and 60 h were replicated three times.

Statistical Methods Used

1. Microsoft Excel used for normalization.
2. ScanArray lite scanner.
3. QuantArray software.
4. GeneSpring
5. Used 3 controls for each DNA spot and

Public Data from Experiment

• Available for Download at the following website [data]

Results from Figures

• Figure 1A:
  • a 2D hierarchical cluster analysis of microarray data. The data is from the BY4743 diploid cell incubated at low temp.
  • A, B, and C in the x-axis represent the LCR genes, whereas E and D represent the ECR genes. **Y-axis shows time going from minutes to hours.
  • Top dendrogram represents similarities in gene expression patterns as the left dendrogram on shows similarities in time exposure to the cold environment.
  • Down-regulation is represented by green, up-regulation is represented by red.

• Figure 1B:
  • shows classifications of ECR genes.
  • The x-axis shows types of functions
  • The Y-axis shows the number of genes

• Figure 1C:
  • Shows classifications of LCR genes.
  • The x-axis shows types of functions
  • The Y-axis shows the number of genes
  • Conclusion: different genes were transcribed by those that regulated cold response over time.

• Figure 2:
  • The transcriptional response as the temperature gets colder from 37°C to 25°C.
  • The X-axis represents temperature change (decrease over time).
  • The Y-axis shows genes that showed a correlation in their transcriptional response as the temperature got lower.
  • Conclusion: as shown in the figure, "a" and "b" were the only genes consistent with previous data and correlated with it as the other genes did not.

• Figure 3:
  • Compares the transactional response to a cold environment and other environmental stresses.
  • Figure 3A shows LCR compared to ESR.
  • Figure 3B shows ECR compared to ESR.
  • The X-axis represents timepoint and type of stress
  • The Y-axis represents Red: Green ratio as it shows levels of gene expression.
  • Conclusion: Opposite transcriptional response between ESR and ECR, however similar gene response between LCR and ESR.

• Figure 4:
  • Cold treatment effect on the regulation of gene expression.
  • The X-axis is time periods from wild-type and msn2/msn4 mutant
  • The Y-axis shows Red: Green ratio.
  • Conclusion: Mutants showed less change in transcription comparing to wild type.

• Figure 5:
  • Accumulation of reserve carbohydrates during cold treatment.
  • The X-axis shows time in hours during the treatment.
  • The Y-axis shows glycogen and trehalose in the cell.
  • conclusion: storage rate of both glycogen and trehalose was greater over time for the wild types than the mutants.

• Figure 6:
  • Transcriptional response in the study compared to another study published in 2002.
    • The X-axis shows time in minutes and hours in both the study and the previous one.
  • The Y-axis shows ribosomal genes.
  • conclusion: similar results in terms of stress response and LCR. ECR ribosomal gene data was different in the two studies.

Evaluation of the Study

• How does this work compare with previous studies?
  • Some of the findings of this study correlates with previous studies and other findings contradicts with previous studies.
• What are the important implications of this work?
  • The important implications of this work is that it gives an idea of how yeast cells response to cold environment and therefore could be related to other cells as yeast cells are model organisms.
• What future directions should the author take?
  • The author should do a study to explore the reasoning that they had some contradicting results with other previous studies in order to prove the "right," "actual" result.
• Give a critical evaluation of how well you think the authors supported their conclusion with the data they showed. Are there any major flaws in the paper?
  • I don't think there were major flaws but the methods could've been written more clearly in terms of the statistical methods. I found it a little confusing as the way it's written was not by stating each method and what they used it for but instead by stating one method multiple times over the article and every time it was stated, different use of it was stated.

Acknowledgments

• I worked with my homework partner, Desiree Gonzalez over text in this project.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Falghane (talk) 21:34, 6 February 2019 (PST)

Refrences

• Customers. (2014, May 12). Main Page. Retrieved from https://www.biology-online.org/dictionary/Main_Page

• Dahlquist, K. & Fitzpatrick, B. (2019). "BIOL388/S19: Week 3 Assignment Page". Week 3 Assignment Instructions

• Schade, B., Jansen, G., Whiteway, M., Entian, K. D., & Thomas, D. Y. (2004). Cold adaptation in budding yeast. Molecular biology of the cell, 15(12), 5492-5502. DOI: 10.1091/mbc.e04-03-0167