Desireegonzalez Week 9

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Purpose

  • The purpose of this weekly assignment is to read and understand how to interpret a scientific research paper on an experiment that is similar in subject to the response to cold shock experiment that we have been working on in class for several weeks. This assignment will assist in determining the proper ways to interpret data and experimental methods presented by other researchers; the data to be interpreted includes graphical representations of data as figures found within the scientific article.

Biological Terms and Definitions

  1. Transcriptome: The full complement of RNA transcripts of the genes of a cell or organism. The types and relative abundance of different transcripts, i.e. the messenger RNAs (mRNAs), can be obtained by analysing cell contents using oligonucleotide microarrays. Such an analysis provides a ‘snapshot’ of the expression pattern of the cell's genes (Oxford Reference 2017).
  2. Inositol: A compound, similar to a hexose sugar, that is a constituent of some cell phospholipids. Inositol is present in many foods, in particular in the bran of cereal grain. It is sometimes classified as a vitamin but it can be synthesized by most animals and there is no evidence that it is an essential nutrient in humans (Oxford Reference 2017).
  3. Oleate: A salt or ester of oleic acid (Oxford Dictionaries 2019).
  4. Chemostat: An apparatus allowing the continuous cultivation of bacterial populations in a constant, competitive environment. Bacteria compete for a limiting nutrient in the medium. The medium is slowly added to the culture, and used medium plus bacteria are siphoned off at the same rate. The concentration of the limiting nutrient in the fresh medium determines the density of the steady-state population, and the rate at which the medium is pumped into the chemostat determines the bacterial growth rate. In chemostat experiments, environmental variables can be changed, one by one, to ascertain how these affect natural selection, or the environment can be held constant and the differential fitness of two mutations can be evaluated (Oxford Reference 2017).
  5. Ergosterol: A plant sterol that, when irradiated with ultraviolet light, is converted to ergocalciferol (vitamin D2). See vitamin D (Oxford Reference 2017).
  6. Trehalose: Mushroom sugar, also called mycose, a disaccharide of glucose. Found in some fungi (Amanita spp.), manna, and some insects; hydrolysed by the intestinal enzyme trehalase (Oxford Reference 2017).
  7. Nucleolar: Relating to a small dense spherical structure in the nucleus of a cell during interphase; for example a nucleolar protein (Oxford Dictionaries 2019).
  8. Translocation: A change in the arrangement of genetic material, altering the location of a chromosome segment. The most common forms of translocations are reciprocal, involving the exchange of chromosome segments between two non-homologous chromosomes. A chromosomal segment may also move to a new location within the same chromosome or in a different chromosome, without reciprocal exchange; these kinds of translocations are sometimes called transpositions (Oxford Reference 2017).
  9. Sphingolipid: A structural lipid in which an acyl chain is attached to sphingosine to form a ceramide. Further modification (glycosylation) gives rise to cerebrosides and gangliosides, major glycolipids in the nervous system (Oxford Reference 2017).
  10. Orthologous: Designating genes (in different genomes) that are derived from a common ancestral sequence (homologous) by phylogenetic descent; related in this way. Also (of a protein or other gene product): encoded by such a gene, or by the same gene in different individuals (Oxford Dictionaries 2019).

Article Outline

Background Information

  1. What is the importance or significance of this work?
    • Temperature affects have been seen to affect enzyme kinetics and cellular processes.
    • For example, suboptimal temperatures have been seen to cause variety in growth, respiration, and lipid membrane composition.
    • Changes in environmental conditions have been seen to trigger stress-response adaptation.
      • An early cold response(ECR) and late cold response have been studied.
  2. What were the limitations in previous studies that led them to perform this work?
    • The limitations that were seen in previous studies was the length of exposure to suboptimal temperatures.
    • The researchers in this article wanted to prolong exposure to stimuli (nonlethal ones) to see how it affected acclimation.

Experimental Procedures

  1. How did they treat the yeast cells (what experiment were they doing?)
    • The researchers treated the yeast cells with a steady state chemostat experiment.
      • Steady state means that all concentrations of metabolites and substrates were constant in time.
  2. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
    • The researchers used the haploid yeast strain, CEN.PK113-7D.
  3. What media did they grow them in? What temperature? What type of incubator? For how long?
    • The researchers grew the yeast strains in anaerobic chemostat cultures with a growth rate of 0.03 per hour, for 12 hours.
    • They used glucose-limited and ammonium-limited chemostat cultures.
    • The chemostat machine used, was 2L with a 1L working volume, a pH of 5, and a stirrer set at 600rpm.
    • The temperatures used for growth were 12 degrees C and 13 degrees C.
  4. What controls did they use?
    • The controls used in the experiment were other researchers data; they used microarray data from various papers as a comparison to see what should be occurring.
  5. How many replicates did they perform per treatment or timepoint?
    • 3 replicates were performed for trehalose per each chemostat.
    • 2 replicates were performed for glucose per each chemostat.
    • 3 replicates were performed for each growth condition.
  6. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
    • To prepare the RNA to the microarray, the researchers of this paper followed the work of Piper et al. (2002); see page 5101 of the article under microarray analysis.

Mathematical/ Statistical Methods Used

  1. What mathematical/statistical method did they use to analyze the data?
    • High performance liquid chromatography using an AMINEX HPX-87H ion exchange column was used to analyze the concentrations of metabolites.
    • DRLANGE cuvette tests were performed to analyze the residual ammonium concentrations.
    • Microsoft Excel was used to find the significance analysis of the microarray data.
    • Venn diagrams and heat maps were used to visualize data generated with Expressionist Analyst Version 3.2.
    • Biomass was analyzed using the Carlo Erba elemental analyzer.
    • The UV method depicted in the Roche kit no. 0716251 was used to analyze the breakdown of trehalose.
    • The Agilent 2100 Bioanalyzer was used to determine RNA quality.
    • Promoter analysis was performed using Regulatory Sequence Analysis Tools (RSA).
    • DAVID (Database for Analysis, Visualiztion, and Integrated Discovery) helped to achieve the GO biological process categories.

Data Collected

  1. Are the data publicly available for download? From which web site?
    • The data (for the microarrays) is publicly available for download.
    • It can be found at the Genome Expression Omnibus Database website below, under the following series number: GSE6190.

Results Presented

  1. What is the main result presented in this paper?
    • The main result presented in this paper is the fact that comparison of chemostat experiments with batch experiments results in large differences between the transcriptional reprogramming and responses seen during long exposure to low temperature acclimation.

Explanation of Figures in the Paper

  1. Table 1:
    • Depicts values for the physiological characteristics of the yeast strain grown in ammonium-limited and glucose-limited anaerobic chemostat cultures.
    • How were the measurements made?
      • The measurements were made by growing media limited cultures, anaerobically at 12 and 30 degrees C, and then using the statistical analysis methods depicted earlier in the outline to obtain values for each physiological characteristic.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The trends shown by the table, are relatively close values for all characteristics.
        • This table can assist in the conclusion that growth efficiency is not severely affected by the growth temperature.
  2. Figure 1:
    • Depicts a Venn Diagram comparing significant differentially expressed genes in N-limited and C-limited chemostat cultures.
    • How were the measurements made?
      • The measurements were made by growing media limited cultures, anaerobically at 12 and 30 degrees C, and then using the Venn Diagram statistical analysis methods depicted earlier in the outline to obtain values for each culture.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The Venn diagram depicts that the N-limited and C-limited cultures have 235 genes deemed significant in common. The N-limited culture also has a higher amount of significant genes compared to the C-limited culture (571>259).
  3. Figure 2:
    • Depicts a heat map representing the transcript ratio of differentially expressed genes in glucose and ammonium-limited chemostat cultures.
      • The measurements were made by growing media limited cultures, anaerobically at 12 and 30 degrees C, and then using the statistical analysis methods depicted earlier in the outline to obtain values for each culture.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The figure depicts that there were 96 genes up regulated at low temperature for both C-limited and N-limited cultures.
      • The figure also shows that there were 139 genes down regulated at low temperature for both C-limited and N-limited cultures.
  4. Table 2:
    • Depicts values for protein storage carbohydrates in biomass of ammonium-limited and glucose-limited anaerobic chemostat cultures.
    • How were the measurements made?
      • The measurements were made by growing media limited cultures, anaerobically at 12 and 30 degrees C, and then using the statistical analysis methods depicted earlier in the outline to obtain values for each culture.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The <0.005 values at 12 degrees C for trehalose and glycogen depict a statistical difference in levels of protein and storage carbohydrates compared to the values at 30 degrees C.
  5. Table 3:
    • Depicts significantly overrepresented cis-regulatory binding motifs and significantly overexpressed promoter elements according to ChiP-on-chip analysis.
    • How were the measurements made?
      • The measurements were made by growing media limited cultures, anaerobically at 12 and 30 degrees C, and then using the statistical analysis methods depicted earlier in the outline to obtain values for each culture.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • Reduced transcript levels at 12 degrees C in nitrogen-limited cultures showed a higher level of STRE elements.
      • This was consistent to the inducing of glycogen and trehalose due to stress from environmental changes in temperature.
  6. Figure 3:
    • Depicts a Venn Diagram and a heat map showing the common genes and their regulation in a three batch-culture study.
    • How were the measurements made?
      • The measurements were obtained from previous studies on the subject of low temperature adaptation.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The variance in the number of genes in each circle of the Venn diagram shows large changes in the response of the genes in the study; this figure helped to depict that gene response (as up or down regulation) was not always consistent.
  7. Figure 4:
    • Depicts a Venn Diagram and a heat map showing common genes and their transcript ratios in a three batch-culture study.
    • How were the measurements made?
      • The measurements were obtained from previous studies on the subject of low temperature adaptation; the data was from transcriptome datasets.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • The number of genes in the Venn Diagram depict that 29 genes were regulated transcriptionally during adaptation and acclimation; however only 11 genes showed a consistent pattern of regulation in all situations.
  8. Figure 5:
    • Depicts four Venn Diagrams comparing the up or down regulation of growth rate dependent genes seen to occur during acclimation or adaptation.
    • How were the measurements made?
      • The measurements were obtained from the comparison of this study's data and previous study's data on the subject of low temperature adaptation.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • Consistent transcriptional responses were seen to occur; an overlap of <0.7% of growth rate responsive genes was observed between the two different experiments.
  9. Figure 6:
    • Depicts four Venn Diagrams comparing the up or down regulation of ESR genes seen to occur during acclimation or adaptation.
    • How were the measurements made?
      • The measurements were obtained from the comparison of this study's data and previous study's data on the subject of low temperature adaptation.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • An extensive overlap of about 63% (50% up-regulated and 13% down regulated) of the genes were consistent in their response although two different experiment types were being used.

Future Directions of the Research

  1. How does this work compare with previous studies?
    • The work compares with other studies by having some consistent gene regulations as previous batch-studies.
    • This work contrasts previous studies since its cultures are providing a flexible and reproducible platform of studies on gene expression in fully acclimatized cultures.
  2. What are the important implications of this work?
    • An important implication of this work is the finding that acclimatized growth at low temperature does not involve the Msn2/Msn4-complex regulatory role; this study suggests that an unknown mode of different regulation was identified.
  3. What future directions should the authors take?
    • The authors should keep expanding the exposure time of the yeast to see if a longer time scale will lead to mutational changes for evolutionary adaptation of the entire genome.

Evaluation of Scientific Article

  1. 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 believe that the authors of this paper did a great job supporting their conclusions with the data they showed. The data on the tables and figures was easy to interpret and was created in a way that made sense when comparing the visualization to the explanation of the data seen in the results and discussion (i.e. the circles in the Venn diagram were proportional to the amount of genes that they were depicting). I also liked how the authors compared their results to the results of other scientific articles, because it helped to create credibility. In addition, I believe that the explanation of discrepancies seen in other scientific articles on the same subject, depicted on page 5101 of the article, helped to show why the researchers were using chemostat experiments instead of batch experiments.

Acknowledgements

  • I communicated with my homework partner, Ava through text message to communicate about how to interpret each of the tables and figures in the research article; we also communicated about how we would be explaining them in our outlines.

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

Desireegonzalez (talk) 23:12, 27 March 2019 (PDT)

References

Chemostat - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095605506 on 26 March 26 2019.

Dahlquist, K. & Fitzpatrick, B.G. (2019, March 26). BIOL388/S19:Week 9. Retrieved from https://openwetware.org/wiki/BIOL388/S19:Week_9 on 26 March 2019.

Ergosterol - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095756465 on 26 March 26 2019.

Inositol - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100004523 on 26 March 26 2019.

Nucleolar | Definition of nucleolar in English by Oxford Dictionaries. (2019). Retrieved from https://en.oxforddictionaries.com/definition/nucleolar on 26 March 26 2019.

Oleate | Definition of oleate in English by Oxford Dictionaries. (2019). Retrieved from https://en.oxforddictionaries.com/definition/oleate on 26 March 26 2019.

Orthologous | Definition of orthologous in English by Oxford Dictionaries. (2019). Retrieved from https://en.oxforddictionaries.com/definition/orthologous on 26 March 26 2019.

Tai, S. L., Daran-Lapujade, P., Walsh, M. C., Pronk, J. T., & Daran, J. (2007). Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis. Molecular Biology of the Cell, 18(12), 5100-5112. doi:10.1091/mbc.e07-02-0131

Transcriptome - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803105320135 on 26 March 26 2019.

Translocation - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803105431114 on 26 March 26 2019.

Trehalose - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803105607273 on 26 March 26 2019.

Sphingolipid - Oxford Reference. (2017, June 16). Retrieved from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100523337 on 26 March 26 2019.



Below are the links to all the Assignments and Journal Entries of the Spring 2019 Semester.

User Page: user:desireegonzalez

Template Page: template:desireegonzalez

Weekly Assignment Pages:

  1. Week 1
  2. Week 2
  3. Week 3
  4. Week 4
  5. Week 5
  6. Week 6
  7. Week 7
  8. Week 9
  9. Week 10
  10. Week 11
  11. Week 12
  12. Week 15

Individual Journal Entry Pages:

  1. desireegonzalez Week 1
  2. desireegonzalez Week 2
  3. desireegonzalez Week 3
  4. desireegonzalez Week 4/5
  5. desireegonzalez Week 6
  6. desireegonzalez Week 7
  7. desireegonzalez Week 9
  8. desireegonzalez Week 10
  9. desireegonzalez Week 11
  10. desireegonzalez Week 12
  11. desireegonzalez Week 15

Shared Journal Pages:

  1. Shared Journal Week 1
  2. Shared Journal Week 2
  3. Shared Journal Week 3
  4. Shared Journal Week 4
  5. Shared Journal Week 5
  6. Shared Journal Week 6
  7. Shared Journal Week 7
  8. Shared Journal Week 9
  9. Shared Journal Week 10
  10. Shared Journal Week 11
  11. Shared Journal Week 12
  12. Shared Journal Week 14/15
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