Angela C Abarquez Week 3

Purpose

The goals of this week's assignment are as follows:

To read a scientific article and evaluate it comprehensively.
To interpret data from a figure and effectively present it at a Journal Club Meeting.
To learn about transcriptional changes in the genes of yeast when exposed to cold-shock.

Journal Club 1

Unknown Term Definitions

Sonicated: Broken cells due to the act of applying sound waves to disrupt particles (biology-online.org, 2009).
Airstream: A current of air (Merriam-Webster).
Trehalose: A disaccharide that is an energy source in some fungi, bacteria, plants and invertebrates (biology-online.org).
Diauxic: The two-phase growth response in microorganism cultures making phenotypic adaptations to the addition of a second substrate (accessscience.com, 2015).
Bactopeptone: An organic nitrogen source in micro-bio culture media (bd.com).
Dendrogram: A diagram with branches representing a hierarchy of categories based on similarities or shared characteristics (Merriam-Webster).
Desaturase: An enzyme that adds double bonds onto the hydrocarbon areas of fatty acids (biology-online.org, 2005).
Ubiquitin: A small regulatory protein in eukaryotes (biology-online.org, 2006).
Glutaredoxin: Reduce disulfides using a dithiol or a monothiol mechanism (Vlamis-Gardikas, A, 2004).
Isoform: Proteins with the same function and a similar amino acid sequence that are encoded by different genes (biology-online.org, 2009).

Article Outline

The following is an outline of "Cold Adaptation in Budding Yeast" by Schade et al., 2004.

Introduction

Unicellular organisms have developed responses to environmental stress
General stress response: a certain set of genes has altered transcription
In environmental stress response (ESR), genes are induced or repressed
- ESR is regulated by the transcription factors Msn2p and Msn4p
One commonly studied example of this is the response of yeast to heat shock, however cold shock has not been extensively studied yet
- A few genes have previously shown to become induced upon cold
The purpose of this article is to analyze the transcriptional changes in cold-shocked S. cerevisiae cells using wild-type cells and those with changes in the Msn2 and Msn4 genes

Materials and Methods

In this experiment, the yeast cells were exposed to cold-shock.

Strains

The BY4743, BSY25 and W303 strains of S. carevisiae were used.

The BY4743 strain, or the wild-type, is diploid.
The BSY25 strain is also diploid and was the product of a cross of two single-mutant strains.
The W303 strain was used for the growth curve and is diploid.

Growth Medium and Culture Conditions

The strains were grown in 50 ml of YPD medium overnight at 30° C. The 50 ml of YPD medium was shaken at 170 rpm in a 250-ml flask.
Next, they were diluted and transferred to a 10° C water bath.
They were then incubated at 170 rpm for either 10, 30, or 120 minutes.
After being harvested, the temperature was decreased by 4° C per minute.

Experimental Design

The strains were harvested at different temperatures, with the controls being those independently grown and harvested at 30° C.
For the wild-type strain, the time points used were:
- 0,2, & 12 hours with two independent biological repeats
- 10 min, 30 min, 60 hours with three independent biological repeats
The other strains (msn2 and msn4) were repeated twice except for at the 12-hour time point (three repeats).
634 genes of the wild-type and 120 genes of the mutant msn2 and msn4 genes were used.
In totality, 43 microarrays were used

RNA Preparation, Labeling, and Hybridization

The hot-phenol method was used to isolate the RNA, with a few modifications.
Reverse transcription was used to label by directly adding Cy3- and Cy5-dCTP.
Hybridization to the microarray:
- The RNA was prehybridized in DigEasyHyb solution, yeast tRNA, and sonicated salmon sperm.
- They were washed twice for 2 minutes in a buffer at 42° C.
- Lastly, they were airstream dried and hybridized.

Data Analysis

The following were used in data analysis:

ScanArray lite scanner
QuantArray software
Normalization using standardized spreadsheets in Microsoft Excel
Normalization of ratios using median ratio of 400 spots
Three quality controls for each DNA spot
Average of log2 values of ratios for duplicate spots
Hierarchical clustering in GeneSpring

The data is available through their website [to Data].

Results

There is a significant overlap between environmental stress response (ESR) and late cold response (LCR)
Carbohydrates (trehalose and glycogen) accumulate during the LCR
Msn2p and Msn4p mediate the LCR
ESR markers were absent in early cold response (ECR)

Main result: The environmental stress response of cold shock in the yeast cells takes place during the late cold response and the early cold response is regulated by a different mechanism.

Figure 1

This figure shows the transcriptional responses to cold measured using the microarray data in 3 ways:

Figure 1A shows an analysis of a time-course experiment with the wild-type through a two-dimensional hierarchical cluster. The green represents down-regulation and red represents up-regulation. On the x-axis, A, B, and C are the LCR genes while D and E are the ECR genes. The y-axis shows the time points of the experiment. The dendrogram at the top shows similarities in gene expression patterns. The dendrogram on the left shows similarities in the amount of time exposed to cold.
Figure 1B shows the distribution of the functional categories in the ECR genes, while Figure 1C shows that of the LCR genes. For these, the x-axis shows the functional categories while the y-axis shows the number of genes.

Measurements were taken from the microarray data and GeneSpring software helped create the clustering.

Findings:

LCR genes are more active than ECR genes
At time point 12, there is a peak in change in transcriptional activity
As the temperature decreases, change in transcriptional activity increases
In the LCR genes, protein synthesis was significantly down-regulated while uncharacterized ORFs were significantly up-regulated

Figure 2

This figure shows a profile of ECR genes over a decrease in temperature from 37 to 25°C. Measurements were obtained from the microarray data at the 2 hour time point in comparison to data from Gasch et al. (2000).

The x-axis shows the decrease in temperature over different time intervals from the 2-hour time point. The y-axis is labeled 'a' and 'b', which represent genes that showed a correlation in their transcriptional response. Red indicates an up-regulation while green indicates a down-regulation.

Findings:

Only clusters a and b were consistent with previous data, however the rest showed inconsistencies.

Figure 3

This figure shows a comparison of transcriptional responses to the cold and other environmental stresses. "A" represents the ECR gene responses compared to those of the LCR genes and other responses. "B" represents the LCR gene responses compared to those of the ECR genes and other responses. The Venn diagrams on the right show the number of genes LCR and ECR have in common with ESR. The microarray data at 2 hours and 12 hours along with data from Gasch et al. (2000) were used for measurement and comparison.

The x-axis shows the time points and the types of stressors. The y-axis shows the red:green ratio abundance.

Findings:

ECR expression contrasts with other stressors (reciprocal transcriptional response)
LCR genes act like ESR genes

Figure 4

This figure shows the expression ratio for gene regulation during cold treatment in the wild-type and msn2/msn4 mutant. The microarray data from 0 hours, 2 hours, and 12 hours was used for measurement.

The x-axis shows the time periods for the wild-type and the msn2/msn4 mutant. The y-axis shows the red:green abundance ratio.

Findings:

Less changes in transcription occurred in the mutants compared to the wild-type
In the ESR, a group of LCR genes were induced in the wild-type but repressed in the mutant

Figure 5

This figure shows the accumulation of reserve carbohydrates, specifically glycogen (top) and trehalose(bottom), in the wild-type strain and msn2 and msn4 mutants during cold shock. At each drop from 30 to 10°C, the glycogen and trehalose levels were measured at the specified time points and used for measurement. Three independent experiments were averaged.

The x-axis shows the time periods in hours during the cold treatment. The y-axis shows the amount of either glycogen or trehalose accumulated in ug glucose/10^7 cells. The light grey represents the wild type strain and the black represents the msn2 and msn4 mutants.

Findings:

No glycogen or trehalose accumulated in the first 2 hours
In the wild type, both carbohydrates increased at 12 hours, and then even more at 60 hours
The mutants only accumulated glycogen after 12 hours

Figure 6

This figure compares the transcriptional responses found to those found in a study by Sahara et al. (2002). "A" shows all of the data while "B" shows a close-up of a cluster that is rich in ribosomal protein encoding genes and "C" shows a close-up of a cluster that was rich in environmental stress genes. GeneSpring program was used to cluster the data from both studies and color code fold changes in transcription abundance.

The x-axis on top shows the time periods of this study and the study by Sahara et al. (2002). The y-axis represents the red:greed abundance ratio.

Findings:

There is a cluster of genes that had a similar response in the LCR in "B" and "C"
In general, the findings of this study contradict those of Sahara et al. (2002)

Discussion

This study was significant because it demonstrates how yeast alter their gene expression, specifically in late cold response, when under cold shock. It also showed that this regulation is both gene and time specific.
There are other mechanisms responsible for the manipulation and regulation of other genes when undergoing environmental stress.
Many previous studies have been conducted on yeast treated with heat, however little work has been done with cold shock so not much was known.
The results of this study are similar to those found in experiments with bacteria through the up-regulation of genes affecting membrane fluidity and mRNA stabilization, however the finding of an early and late cold response is different than what was found in previous work with prokaryotes
In the future, the authors could study the mechanisms behind the early cold response. Another idea would be to test cold shock responses on a eukaryote to see if there are any similarity in the genes that express transcriptional changes.

Critical Evaluation

The study conducted provided plenty of evidence to support the conclusion. I appreciated how they included carbohydrate accumulation and used specific transcription factors that are known to be involved in stress responses. I also think the authors did a great job of tying their findings to other people's work and making important comparisons. The only critique I have is that the figures were difficult to read. It is more difficult to judge a value by dullness of color versus looking at a number. There was also one figure (Figure 1A) that had a label "F" that wasn't mentioned in the description, so it is confusing what this pertains to.

Acknowledgments

I met with my homework partner, Brianna, on Wednesday, February 6 for about 3 hours to discuss the article and review our figure. We started our outlines together and then practiced presenting our figure. I also referenced her Week 3 page for guidance on citations.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Angela C Abarquez (talk) 13:29, 6 February 2019 (PST)

References

AccessScience Editors. (2015). Diauxic growth (diauxie). In AccessScience. McGraw-Hill Education. Retrieved from https://doi.org/10.1036/1097-8542.BR0105151

BactoPeptone. Retrieved from https://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211677.pdf

Biology Online Dictionary. Desaturase. (2005). Biology-online.org. Retrieved from https://www.biology-online.org/dictionary/Desaturase

Biology Online Dictionary. Isoform. (2009). Biology-online.org. Retrieved from https://www.biology-online.org/dictionary/Isoform

Biology Online Dictionary. Sonication. (2009). Biology-online.org. Retrieved from https://www.biology-online.org/dictionary/Sonication

Biology Online Dactionary. Trehalose. Biology-online.org. Retrieved from https://www.biology-online.org/dictionary/Trehalose

Biology Online Dictionary. Ubiquitin. (2006). Biology-online.org. Retrieved from https://www.biology-online.org/dictionary/Ubiquitin

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

Merriam-Webster Dictionary. Airstream. Merriam-webster.com. Retrieved from https://www.merriam-webster.com/dictionary/airstream

Merriam-Webster Dictionary. Dendrogram. Merriam-webster.com. Retrieved from https://www.merriam-webster.com/dictionary/dendrogram

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