Brianna N. Samuels-Week 3

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Goal

  • To read a scientific article and obtain knowledge that I can use in the first Journal Club of the semester
  • To understand what occurs when yeast is introduced to cold shock and what effects it has on transcription

Terms

  • trehalose: a disaccharide made up of two alpha-glucose molecules, and serves as an energy source in certain fungi, bacteria, plants and invertebrates (Biology Online Dictionary, 2014)
  • shine-dalgarno sequence: 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 (Biology Online Dictionary, 2014)
  • Optical Density (Optical Density 600 specifically): the measure of the amount of light absorbed by a suspension of bacterial cells or solution of an organic molecule with the use of a colourimeter or a spectrophotometer (Biology Online Dictionary, 2014) (the 600 represents the wavelength observed)
  • sonicated: Sonication uses sound waves to agitate particles in a solution. It converts an electrical signal into a physical vibration to break substances apart. These disruptions can mix solutions, accelerate the dissolution of a solid into a liquid, such as sugar into water, and remove dissolved gas from liquids. In DNA testing, sonication breaks apart molecules and ruptures cells, releasing proteins for testing. (Sciencing, n.d)
  • DigEasyHyb: a nontoxic, ready-to-use hybridization buffer (Sigma Aldrich, n.d)
  • Tagged Image File Format (TIFF files): a standard file format that is largely used in the publishing and printing industry. The extensible feature of this format allows storage of multiple bitmap images having different pixel depths, which makes it advantageous for image storage needs (Techopedia, n.d)
  • hierarchial clustering: an algorithm that groups similar objects into groups called clusters. The endpoint is a set of clusters, where each cluster is distinct from each other cluster, and the objects within each cluster are broadly similar to each other. (DISPLAYR, 2018)
  • diauxic: the diphasic (two-phase) growth response seen in a culture of microorganisms making a phenotypic adaptation to the addition of a second substrate (Access Science, 1970)
  • transient: short-lived; passing; not permanent; said of a disease or an attack (Biology Online Dictionary, 2014)
  • desaturase: Any of several enzymes that put double bonds into the hydrocarbon areas of fatty acids (Biology Online Dictionary, 2014)

Outline

Introduction

A. Background Info

  • All unicellular cells have programmed responses to stress
    • this is due to drastic changes in the environment constantly subjected to them
    • Long term vs short term stress response
      • long term: transcription can be altered/changed
      • short term: degradation and phosphorylation of the protein

B. Yeast

  • Extensive study of yeast stress
    • introduction of heat shock causes rapid induction of heat shock proteins
      • these proteins cause stability for cellular proteins as well as a reactivation for the heat damaged proteins
    • transcription changes in response to various forms of stress
    • 10% of the genome is repressed and induced when responding to stress
    • ESR= Environment stress response genes
      • when induced, the gene is usually helpful with protein folding/degradation, transport, and carbohydrate metabolism
      • when repressed, the gene is usually used in processes of cell growth
      • regulated by transcription factors Msn2p AND Msn4p

C. What are we looking for?

    • response to cold stress as well as other stress response at a transcriptional level

Materials and Methods

A. Strains Used

  • BY4743
    • Diploid wild type
  • BSY25
    • Diploid cross between mutant strains
  • W303
    • used for growth curve
    • Diploid

B. Growth Conditions

  • medium of choice is YPD (50mL)
  • incubated at 30°C overnight
  • diluted and grown again
  • transferred to 10°C water bath and incubated for 10, 30, or 120 minutes total
    • the temperature would drop every minute by 4°C
  • harvested at 10 or 30°C (control)

C. Experimental Design

  • Wild type strain
    • time course experience: 0 hours, 2 hours, and 12 hours (these were repeated 2 times) as well as 10 minutes, 30 minutes and 60 hours (these were repeated 3 times)
  • Mutant strain
    • time course experience: 0 hours, 2 hours, and 12 hours (these were repeated 2 times except for at 12 hours which was ran three times)
  • 43 microarrays used
  • 120 genes from mutant and 634 from the wild type

D. RNA isolation

  • isolated using a slightly modified version of the hot phenol method
  • reverse transcription was used to label parts of the mRNA which was hybridized to the yeast microarrays
    • this was prehybridized in DigEasyHyb solution, sonicated salmon sperm, and yeast tRNA

E. Data Analysis

  • GeneSpring Software
  • subarray normalization
  • QuantArray Software
  • TIFF files
  • Microsoft Excel
  • averaged log2 values

Results

A. Figures

  1. (A.) the x axis showed the different types of clusters labeled A-F (A-C represent late response and D-F represent the early response) and the y-axis showed the time corresponding. colors represent whether they are induced or repressed (green is down regulation while red is up regulation)The top dendrogram represents similarities in the gene expression and the one on the left represents the similarities in the time it was exposed to cold. The length of the gap showed the amount of activity occurring. the measurements were made using the microarray data and the GeneSpring Software for the hierarchial clusters (B.) represents the early cold response. the x axis represents the most representative types of functional categories and the y axis represents the number of genes. the darker color represents the down regulation and the lighter color represent the up regulation. the measurements were made using the microarray data and the GeneSpring Software for the hierarchial clusters. these as stated earlier are responsible for transcription, transport, and metabolism. (C.) represents the late cold response. the x axis represents the most representative types of functional categories and the y axis represents the number of genes. the darker color represents the down regulation and the lighter color represents the up regulation. the measurements were made using the microarray data and the GeneSpring Software for the hierarchial clusters. these as stated earlier are responsible for protein formation/degradation and metabolism.
  2. the x axis represents time and temperature decrease. the y axis represents the transcription abundance ratio. a and b represent the genes that had a correlation during the temperature shift. it was measured with the microarray data. most of the data showed the opposite effects of the previous data used to compare. only a and b on this figure was similar to the previous data
  3. the x axis represents time and the different types of stress applied. the y axis represents the transcription abundance ratio. it was measured with the microarray data and other data from another source. the late cold response genes response to the other stressors was similar to ESR while the early cold response genes had a reciprocal transcriptional response
  4. the x axis represents the temperature and the control vs the wild type and mutant results. the y axis represents the transcription abundance ratio. they were measured with the microarray data. since 78% of the late cold response genes weren't affected by the missing transcription factors, it was assumed that there are other regulators involved. there wasn't a significant difference in the early cold response genes between the wild type and mutant so it was assumed that it was independent of the transcription factors
  5. the x axis represents time after the cold shock and the type of gene (early cold response vs. late cold response). the y axis represents the amount of trehalose and glycogen measured.the dark grey represents wild type and the black represents the mutant. it was measured in hours on the x axis and microgram/10,000,000 cells and using the average of the microarray data. the mutant strain ended with a small amount of glycogen produced and no trehalose (this corresponds specifically to the late cold response). the wild type ended with a small amount of trehalose and more glycogen.
  6. the x axis represents time. the y axis represents the transcriptional abundance ratio.

Discussion

A. this work compared to others done has been pretty consistent B. important implications of this work

  • we know yeast in response to cold shock change their gene expression
    • we know yeast has an early cold response and a late cold response

C. Further direction

  • maybe the author can dive in to the other types of regulatory mechanisms involved, the posttranslational and translational level

D. Critique

  • I thought the author conveyed what he was trying to convey well. I believe that all of the work done was explained thoroughly and tied together throughout the whole paper. A few times when looking at the figures, I struggled to see what they truly were trying to convey but that could be just my lack of knowledge on how these things work.

Acknowledgements

  • Homework Partner: Angela Abarquez Met face-to-face in LSB and worked on outline together. We also discussed our shared figure being presented
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

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