Sahil Patel Week 3 Assignment

Sahil Patel Electronic Lab Notebook

Purpose

The purpose of this assignment is to prepare ourselves for our first Class Journal club on the article for this week.

Preparation for Journal Club 1

Key Terms for Article

Reverse transcription: The process of making a double stranded DNA molecule from a single stranded RNA template through the enzyme, reverse transcriptase (www.biology-online.org).
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 (www.biology-online.org).
Hybridization: The process of forming a double stranded nucleic acid from joining two complementary strands of DNA or RNA (www.biology-online.org).
Diauxic: The two-phase growth response in microorganism cultures making phenotypic adaptations to the addition of a second substrate. (www.accessscience.com)
Hierarchal 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 (www.displayr.com).
Dendrogram: A diagram, similar to a family tree, that indicates some type of similarity between different organisms (Hine, 2015).
Budding: A method of asexual reproduction in which a new individual is derived from an outgrowth (bud) that becomes detached from the body of the parent (Hine, 2015).
YPD medium: A nutritious medium available in liquid (broth) or solid (agar) forms for the growth and propagation of yeast cultures. It primarily contains of bacteriological peptone, yeast extract, and glucose (www.sigmaaldrich.com).
Ubiquitin: a chiefly eukaryotic protein that when covalently bound to other cellular proteins marks them for proteolytic degradation especially by a proteasome (www.merriam-webster.com/medical)
log Phase: the period of growth of a population of cells in a culture medium during which numbers increase exponentially and which is represented by a part of the growth curve that is a straight line segment if the logarithm of numbers is plotted against time (www.merriam-webster.com/medical).

Article Outline

Introduction

Single-cell organisms face environments with volatile harsh conditions that are ever changing
This article studies the cell's response to its changing environment particularly when experiencing cold shock
- Focused on yeast's ability to adapt to cold treatment through gene expression
- Measured by intensity and duration of cold treatment
- Previous research has focused on heat shock, but not much research in the effect of cold shock on yeast on a molecular scale.

Methods and Procedure

Strain Used

The strains used were the BY4743, BSY25 and W303 strains of S. carevisiae
- BY4743: diploid wild-type
- BSY25: diploid cross between mutants
- W303: diploid (only used for growth curve)

Media Used

Yeast cultures were inoculated in a YPD medium
- Grown overnight at 30 degrees at 170rpm
- They were then diluted and grown again placed in 10 degrees condition
- Then incubated for 10, 30, 120 min as the temperature decreased at 4 degrees per min
- Harvested cells at either 10C (experimental unit) or 30C (control) -- log phase

Controls

The cultures were kept in the standard physiological conditions at 30 degrees Celsius
Data was recorded at same time points for later comparison

RNA Isolation and Hybridization

RNA was isolated via hot-phenol method
Labeled RNA through reverse transcription then purified using the Oligotex Spin-Column Protocol

Data Analysis Techniques Used

Microarray slides scanned using a ScanArray lite scanner.
Hierarchical clustering of genes
GeneSpring software
QuantArray software
Microsoft Excel

Results

Each DNA spot had to pass three quality controls to be included in the normalization and subsequent analysis:

Signal intensity had to be significantly greater than the local background
Signal intensity must be within the dynamic range of the photomultiplier tub
The raw intensities for duplicate spots had to be within 50% of each other

Complete Data Available @: http://cbr-rbc.nrc-cnrc.gc.ca/genetics/cold/

Figures

Figure 1a: Two-dimensional hierarchical cluster analysis of microarray data
- x-axis: LCR (A,B,C) or ECR (D,E) gene that is either down regulated (green) or up regulated (red)
- y-axis: Duration that cells were exposed to cold environment
- Methods: Measurements were made using microarray data and clustering was done through the GeneSpring software.
- Results: This figure showed that genes did respond to the cold and induce transcription. Statistically significant variation was found of at least twofold on some of the 634 genes analyzed. Figure was vague as to what letter F represented; was not specified
Figure 1b: Classification of ECR genes
- x-axis: Cell functions
- y-axis: The number of genes involved in the ECR
- Methods: Data was analyzed on MIPS classification and the SGD database
- Results: The genes that respond to the cold temperature transcribe different genes with their own respective functions based off of the duration of the cold treatment.
Figure 1c: Classification of LCR genes
- x-axis: Cell functions
- y-axis: The number of genes involved in the LCR
- Methods: Data was analyzed with MIPS classification and the SGD database
- Results: The genes that respond to the cold temperature transcribe different genes with their own respective functions based off of the duration of the cold treatment.
Figure 2: Transcriptional profiles of early cold response during temperature downshifts
- x-axis:Duration that cells were exposed to cold environment
- y-axis: Red(Induced):Green(Repressed) ratio during transcription
- Methods: Ratios of red:green found through the use of microarrays compared with the microarray data of Gasch et al., 2000
- Results: Some similarities in the resulting clusters from this study in comparison to the previous study done by Gasch however some expression patterns appear to be different
Figure 3 (A,B,C): Comparison of the transcriptional responses to cold and other environmental stressors
- x-axis: Duration stressor was applied and distinction among the different stressors tested
- y-axis: Transcription abundance ratio (Red:Green)
- Methods: Use GeneSpring to compare with cold treatment data
- Results: ECR genes show a reciprocal transcriptional response while LCR genes showed similar gene expression when compared to other ESR.
Figure 4: Regulation of gene expression during cold treatment
- x-axis: Time and Strain type
- y-axis: Transcription abundance ratio (Red:Green)
- Methods: Gene expression ratios averaged from these points: 0hr (30C), 2hr (10C), 12hr (10C)
- Results: Less change in rate of transcription in the mutant types compared to the wild type
Figure 5: Accumulation of reserve carbohydrates during cold treatment
- x-axis: Duration of cold treatment
- y-axis: Levels of glycogen or trehalose present in cells
- Methods: The levels of glycogen and trehalose were averaged from three treatments at several time intervals after temperature shift from 30 to 10°C
- Results: Wild types showed an increase in glycogen as well as trehalose particularly by the 12th hour of cold treatment. Glycogen and trehalose levels in the wild type strains were far greater than those with mutated genes showing the importance of the carbohydrate storage gene.
Figure 6: Comparison of results obtained in this study with that of Sahara et al. (2002)
- x-axis: Time passed for each study
- y-axis: Transcription abundance ratio (Red:Green)
- Methods: Using GeneSpring, the transcriptional profiles were clustered and analyzed
- Results: Significant overlap between the LCR and ESR genes indicating that the ESR is activated during the LCR. Comparison with the ECR revealed very different expression profiles suggesting a reciprocal stress response during the first 2hr of cold treatment.

Discussion and Final Thoughts

The transcriptional response of S. cerevisiae when exposed to the cold treatment had two varying results during the early and late phases
- During the early phase, the cell was able to maintain transcriptional efficiency by adjusting its membrane fluidity and through the destabilization of RNA secondary structures.
- During the late phase, it was observed that the cell induced genes that encode for proteins that are involved in protecting it from general stressors.
This study helped us see how the cell adapts and controls its gene expression when exposed to, in this case, the cold shock; whereas before this study most research had only been done with a cell's response to heat shock or other environmental stressors.
I agree with the authors in their step forward where they suggested that the next focus should be on the key regulatory mechanisms that allow cells to survive and grow in the cold.
I was thoroughly convinced with the way the author presented their study. The case started with good background information and a well organized experiment and the data and results were well expressed through the figures throughout the article.

Acknowledgments

I would like to thank my homework partner for this week Leanne K. Kuwahara for helping me in interpreting certain figures in the assigned article via text.

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

Sahil Patel (talk) 08:15, 7 February 2019 (PST)