Alondra Vega: Week 9

Vocabulary

1. Doubling time:the time taken for a cell to complete the cell cycle. The amount of time between successful cell divisions.[1]
2. Reverse transcription:The process of making a double stranded DNA molecule from a single stranded RNA template through the enzyme, reverse transcriptase.[2]
3. Heat shock:The response is mediated by heat shock transcription factor (HSF), which is present in a monomeric, non-DNA binding form is unstressed cells and is activated by stress to a tromeric form which can bind to promoters of heat shock genes.[3]
4. Glycogen:A branched polymer of glucose that is mainly produced in liver and muscle cells, and functions as secondary long-term energy storage in animal cells.[4]
5. Trehalose:A sweet-tasting, crystalline disaccharide, C12H22O11, found in trehala and in many fungi.[5]
6. Osmotic Stress: The osmotic stress technique is a method for measuring the effect of water on biological molecules, particularly enzymes. Just as the properties of molecules can depend on the presence of salts, pH, and temperature, they can depend significantly on the amount of water present.[6]
7. Hyperosmotic:Of, relating to, or characterized by an increased osmotic pressure (typically higher than the physiological level).[7]
8. Osmolarity: The molarity of an ideal solution of a nondissociating substance that exerts the same osmotic pressure as the solution being considered. [8]
9. Dendrogram:a branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics especially in biological taxonomy.[9]
10. Biogenesis:The process in which life forms arise from similar life forms.[10]

Outline: Cold Adaptation in Budding Yeast; Schade et al.

Abstract

• The paper focused on determining the transcriptional response to a stress, specifically cold shock, in budding yeast.
• Microarrays were used to check the transcript abundance, which clearly showed two different groups of transcriptionally modulated genes.
  • Early cold response (ECR)
  • Late cold response (LCR)
• Comparisons were made to other stresses.
• Trehalose and glycogen are induced in the LCR, which means that the environmental stress response (ESR) happens during the LCR.
• Msn2p and Msn4p are involved in induction of genes and they control the stress response in the LCR.
• Cold-specific early response is not mediated by Msn2p and Msn4p, but by a unknown regulatory mechanism.

Introduction

• Unicellular organisms can be exposed to changes such as changes in nutrients, acidity, osmolarity, temperature and exposure to toxic agents and radiation.
• Cells have developed a way to adapt to these changes, whether it be by protein phosphorylation and degradation or by transcriptional changes.
• Previous research in heat shock shows that the mediated transcription factor in this process is HSF1p.
• Genome-wide transcriptional profiling has shown that approximately 10% of the genes are induced and/or repressed in the response to a specific stress. The genes involved are called the environmental stress response (ESR).
• Repressed ESR genes can be found in processes such as RNA metabolism, nucleotide biosynthesis, secretion and ribosomal performance.
• The regulation of the ESR genes is determined by two distinct transcription factors, Msn2p and Msn4p.
• Cold shock has not been studied as much as the other stresses, which is a motive for this research. Researchers know that cold causes changes in the physical and biochemical properties of the cell.
  • An example of this is a decrease in membrane fluidity, which will result in slower lateral diffusion of membrane proteins.
• Cold shock in E. coli is described.
• Previous research shows that in yeast, differential hybridization revealed a set of genes that were up-regulated during a cold shock: NSR1, TIP1, TIR1, TIR2 and NSR1. These genes have been fund to be part of pre-rRNA processing and ribosome biogenesis.
• TIR1, TIP1, and TIR2 are responsible for maintaing the cell wall integrity during stress.
• This article is focused on describing the global transcriptional analysis in the stress cold shock in yeast. From their data, they were able to compare their results to other stresses to measure the general stress response as well as the trehalose and glycogen levels.

Materials and Methods

Strains Used

• The Schade lab used strains BY4743(wild type) and BSY25. These are diploid. These are a result of a cross of two single-mutant strains from the American Type Culture Collection.
• W303 was also used for growth curve experiments, this is a haploid.
  • Note: Knowing the how and where the Schade Lab got their strains is important when trying to replicate this experiment.

Growth Medium and Culture Condition

• Yeast were grown in YPD medium, which contains glucose bactopeptone and yeast extract.
• Cultures were inoculated and grown overnight at 30°C in 50mL of medium.
• The cultures were then diluted in fresh medium, grown to have a concentration of 0.6 OD₆₀₀ at 30°C and the transferred to a 10°C water bath shaker.
• They cells were incubated for 10, 30 and 120 minutes at 170rpm before harvesting.
• The temperature then decreased 4°C per minute.
• Doubling time was determined to be 20.7 hours. Based on this observation, the diluted cultures for the 12hour experiment were only allowed to reach a concentration of 0.4 OD₆₀₀ before they were changed to the 10°C flask.
• The 60hour experiments were diluted to 0.05 OD₆₀₀, it was not until the cells reached a concentration of 0.1OD₆₀₀ that they were then moved to the 10°C flask.
• All cultures reached a final OD₆₀₀ of 0.6-0.8 before harvesting the cells at 10°C. Cell platelets were frozen in liquid nitrogen and stored at -80°C.
  • Note: The control for this experiment was harvested at 30°C.

Isolation of RNA

• Total RNA was isolated form the cells using the hot phenol method, which can be found in the Kohrer and Domney 1991 paper, with some modifications.
• Cells from a 500mL culture were processed by extracting phenol twice for 10 minutes apiece.
• For the 60-hr experiment, RNA isolation was shown to be inefficient, thus to make this process easier and actually retrieve some RNA, glass beads were added.
• Also, for their mRNA purification the Oligotex Spin-Column Protocol was used.
  • Note: The Oligotex Spin-Column Protocol is a Kit that can be purchased that come with its own protocol and materials to purify the mRNA.

RNA labeling and DNA Microarray Hybridization

• Three micrograms of mRNA were labeled by incorporating Cy3 and Cy5 through reverse transcription.
• The cDNA that was retrieved from the previous step was hybridized onto yeast genomic DNA microarrays.
• Prehybridization was done 20:1:1.
• Microarray were washed twice in 01XSSC buffer for 2min/wash at 42°C, airstream dried, and immediately hybridized.

Data Acquisition and Analysis

• Microarrays were scanned using a lte scanner at a 1-μm resolution and the TIFF files were quantified using the QuantArray software.
• Normalization and quality controls were all done in an Excel sheet.
• Each DNA spot had to pass three quality controls.
  • The signal intensity had to be much greater than the background intensity.
  • The signal intensity had to be between the dynamic range of the photomultiplier tube.
  • The raw intensities of the duplicate spots had to be within 50% within each other.
• If the spots met all three criterion, then the ratios of the intensities were normalized by the median ratio of the entire subarray. In this case the subarray consisted of 400 spots.
• Each subarray was normalized individually, which yielded more reproducible data.
• The log2 values for each duplicate were averaged.
• Statistical Analysis and visualization were performed with GeneSpring software.

Experimental Design

• Samples were taken before harvesting to determine the budding index and the glucose content in the medium.
• On averaged there were about 70% budded cells and the medium glucose content was 16g/L.
• The 12-h and 60-h experiment were tested for diauxic shift-inducible genes.
• There were three time course experiments with the wild type strain that were performed with three time points, 0,2 and 12hours. There were two repeats. for the experiments in the times of 10 min, 30 min, and 6 hours, there were three repeats.
• For each experiment performed the Cy dye was changed for the reference and experimental samples.
• The control also had three replicates with the dye swapping.
• From the control hybridization, data was obtained from 5559 genes and about 14 genes showed an average variation >1.5 fold.
• Genes with twofold variation were selected.
• Student T-test was used with a p-value of <0.03 for the experimental analysis.
• The expression ratios were averaged.
• A total of 43 microarrays were used.

Comparison with Other S. cerevisiae Stress Data

• this section of the methods section of the paper describes were they got all their comparison data. The ESR genes and the study of the response to variety of stresses were obtained from different websites. The data for cold shock was obtained from the Sahara et al. 2002 database. All comparisons were done through GeneSpring, which is a program, using standard correlation.

Biochemical and Analytical Procedures

• To find the trehalose and glycogen levels, they looked at the Parrou and Francois 1997 paper.
• For this experiment, the cells were grown and harvested as they were in the DNA microarray analysis.
• Glucose levels were determined by using the Glucose kit.

Results

• When grown at 10°C, yeast cells showed a reduced growth rate but a normal growth curve. All this means that the ceels grew how they were supposed to, just took longer than usual to get to a certain point.
• After 120 hours the cells reached stationary phase showing low glucose concentrations.
• An important factor to keep in mind was membrane fluidity. Previous studies have identified cold-inducible fatty acids desaturases.
  • Schade's lab was interested in identifying the genes that were involved in lipid metabolism and from there check which ones were responsible for membrane fluidity.
    • What they found was that UFD1 was induced during the ECR. Other genes that showed a similar affect during the ECC never exceeded twofold.
• A set of induced LCR genes were found that encode proteins that are known to be involved in stress response. They have been shown to prevent protein aggregation and facilitate protein degradation or refolding.
• Genes shown previously to be induced in oxidative stress were also induced in the LCR.
• They compared the ECR and LCR transcription profiles to different environmental stresses.
• They chose to compare the 2hour and the 12 hour time points because those showed the max changes in transcript abundance.
• Schade's Lab was interested to see whether transcription factors are involved in the regulation of cold responsive genes through microarray analysis.
  • Cold exposure showed no detectable lag in growth compared to the wild-type strain.
  • When looking at cold-induced genes dependent on Msn2p and Msn4p showed no activation in the mutant strain. They also occur with less intensity when compared to the wildtype strain. In comparison to cold-repressed Msn2p/Msn4p-dependent genes show an increase in the signal intensity.

Figure 1

• • Their results showed that yeast cells fail to respond to a rapid temperature change from 30-10°C. This means that gene expression did not change too much during the early stages of the experiment.
  • They were able to group the genes into five main clusters: 3 cluters of induced genes and 2 clusters of repressed genes.
  • There was a set of genes that were induced during the first 2 hours of the experiment, shown as clusters D and E in the first part of the figure. The other subset was induced or repressed after 12 and 60 hours, which are cluster A-C.
  • Clusters D and E are considered the ECR and clusters A-C are defined as LCR.
  • The genes had higher expression levels during the LRC, specifically during the 12 hour peak.
  • Clusters D and E were induced at least 2-fold and identified 130 open reading frames. The genes found in these clusters are are associated with transport, lipid and amino acid metabolism and transcription.
    • They found a set of ECR genes involved in transcription, including the RNA helicases genes, which goes along with previous studies made.
  • Cluster F shows the genes that had their expression reduced during the ECR, specifically in the first 2 hour period. This cluster includes the heat shock proteins , which are required to have proper protein folding and who have shown to play an importnat role in response to stress.
  • They identified 280 LCR genes that were induced twofold or more, which can be seen in Cluster C.
    • These genes included the genes that are responsible for carbohydrate metabolism, specially glycolysis, glycogen metabolism, and trehalose metabolism.
  • The long-term cold treatment repressed a variety of genes, and 256 cold-repressible genes were found. These can be found in clusters A and B.
  • Cluster A shows the genes that are mainly involved in protein synthesis.
  • Cluster B shows genes that are associated with nucleotide biosynthesis, protein modification and vesicle transport.
  • The results shown in this figure suggest that repression of ribosomal genes and genes involved in protein synthesis contribute to the adaptation to cold in yeast.
  • Parts B and C of this figure show how the ECR and LCR genes were classified into functional categories according to MIPS. The diagrams just show the distribution of the genes into the respected areas. Also, each category is divided into up-regulated and down-regulated genes. In parentheses we can see the total number of genes classified in each category according to MIPS.
  • Important Note: Part A in Figure 1 shows the down-regulated genes in green and the up-regulated genes in red.

Figure 2

• • Focuses on showing the comparison of ECR with transcriptional patterns produced from a temperature chnage of 12°C (37-25°C).
  • Forty-seven percent showed a transient increase in expression after a shift from 37-25°C.
  • Cluster B contains the genes that are involved in transcription and in amino acid and fatty acid metabolism.
  • Cluster A shows that the majority of repressed ECR genes were also repressed during this temperature shift.
  • Weak correlations were seen when comparing transcriptional profiles from cultures at low temperatures (15, 17, or 21°C).
  • Important Note: The temperature downshift that was compared to the ECR genes, had different time points that were looked at in this figure. they were 5, 15, 30, 60, and 90 minutes.

Figure 3

• • Focuses on showing on how the ECR expression profiles compare to other stresses such as oxidative stress, osmotic stress, disulfide reducing agent and heat shock, which showed unexpected correlations for osmotic stress.
  • Part A of this figure shows how ECR genes had reciprocal behavior under other stress stimuli. Genes that were induced in ECr were repressed in other stresses and vice versa.
  • Cluster I shows that almost half of the repressed genes in ECR were induced during heat shock, which include the heat shock proteins.
  • Cluster II shows that 40% of the induced ECR genes that were repressed after 0.5 hours of heat shock. These genes deal with RNA metabolism.
  • Few genes were coexpressed.
  • Part B of this figure shows the expression profiles of the LCR along seen with other stresses. They observed similar transcriptional responses in all cases.
  • There is a reciprocal response pattern in the ECR, which reverts back to the general stress response during LCR.
  • Part C, the top venn diagram compares the similarities between the induced LCR genes and the ESR. They found that there was a significant overlap of 87 genes, which included the genes HSP12, HSP104 and the ones involved in carbohydrate metabolism.
  • Part C, second to top shows the similarity between the repressed genes and the ESR, which showed a significant overlap of 111 genes. This consisted of genes inviolved in nucleotide biosynthesis and ribosomal genes.
  • Part C, bottom two diagrams show the similarities between ESR and ECR genes. There was no significant overlap , as a matter of fact there were only two genes that were induced and two genes that were repressed.
  • The data shown in this figure suggests that the LCR involves the ESR, on the other hand the ECR gives a "cold-specific" transcriptional response.

Figure 4

• Shows how the relative expression of 120 genes were affected >twofold in the Δmsn2 Δmsn4 starin when exposed to cold.
• Msn2p and Msn4p were both required for the activation of 99 LCR genes. These are shown in the cluster that is labeled ESR.
• At 0 hours, a few genes were observed that required Msn4p and Msn2p at 30°C.
• About 78% of the LCR genes were unaffected by the absence of Msn2p and Msn4p, which may mean that there must be additional transcriptional regulator for LCR gene expression.
• The 2 hour cold treatment did not reveal significant differences in transcript abundance between the wild-type and the two strains. This means that Msn2p and Msn4p are independent regulation of ECR genes.

Figure 5

• Shows that during the first two hours of the experiment there is no accumulation of glycogen and trehalose.
• There was an increase in glycogen and trehalose content was observed after 12 hours of the experiment.
• The results in this figure are in agreement with the microarray data, which says that carbohydrate metabolism is induced during the LCR.
• In the mutant strain there was a small accumulation of glycogen and no accumulation of trehalose occurred in the LCR when exposed to cold temperatures.
• The data shows the connection with the loss of induction of these particular genes during cold shock in the Δmsn2 Δmsn4 strain.

Discussion

• Yeast gives different expression programs in response to the cold and their regulation is time and gene specific.
• The ECR is responsible for the induction of genes in RNA metabolism and lipid metabolism, while the genes that are induced in the LCR are responsible for mainly encoding proteins that are involved in protecting the cell against the environmental stresses.
• Genes that encode cold-induced genes encoding RNA helicases, RNA-binding proteins, and RNA-processing proteins during the ECR.
• RNA helicases are important during cold adaptation.
• They observed accumulation of both carbohydrates during the LCr, which confirms the observed induction of the genes involved in this particular synthesis.
• There is previous data that suggests that trehalose is an important component of the cold-adaptation process. Unfortunately, the Schade lab was not able to detect a decrease in growth rate or viability in the strains studied.
• There were some HSP genes that were found to be induced in the LCR, which means that there is a need for protein folding and maintaining protein conformation in the cold.
• Proteins that cannot be be folded properly will undergo degradation, which are also induced during the LCR.
• They mention in the paper how difficult it can be to compared their data with another lab's data because of all the different variables.
• Comparison to the Gasch et al. paper showed an overlap between LCR and ESr genes. This means that the environmental stress response is activated during the LCR.
• Comparing it to the ECR, there was a reciprocal stress response during the first two hours of cold adaptation.
• The stress induction during the LCR is controlled by the Msn2p/Msn4p transcription factors.
• The regulation of LCr repressed and induced genes has been shown to be dependent of protein kinase A pathway in response to carbon source and on protein kinase C pathway.
• The ECR transcriptional pattern was unchanged in Δmsn2 Δmsn4 double mutant.
• This shows that a cold-specific mechanism for the ECR, which may involve an uncharacterized regulatory factors.

Figure 6

• They compared their data to the Sahara data that was published in 2002.
• Parts B and C show the common cluster of genes during the LCR, which includes general stress response genes.
• Parts A and B show the major differences between the tw data sets.
  • An example is that Sahara showed induction of ribosomal genes during short cold treatments, while they observed a decrease in abundance for ribosomal genes.

Conclusion

• There are two different expression phases: early response and late response.
• The early phase aids with adjustments of membrane fluidity and destabilization of RNA secondary structures that will allow efficient protein translation.
• The late phase aids with the environmental stress response which by consequence alters the accumulation of misfolded proteins and reduced enzyme activity.
• The transcriptional response to cold involves both general stress and and cold-specific mechanisms.
• Future experiments are needed to find the key regulatory mechanism that will allow cells to survive and grow in the cold.

Alondra Vega

BIOL398-01/S11:Assignments

Lab Journal

Class Journal Week 1	Shared Journal: Week 5	Shared Journal: Week 9
Class Journal Week 2	Shared Journal: Week 6	Alondra Vega: Week 10	Shared Journal: Week 13
Class Journal Week 3	Alondra Vega: Week 7	Shared Journal: Week 11	Shared Journal: Week 14
Shared Journal: Week 4	Shared Journal: Week 8	Shared Journal: Week 12	Alondra Vega:Week 15

Individual Assignments

Alondra Vega: Week 2	Alondra Vega: Week 6	Alondra Vega: Week 11
Alondra Vega: Week 3	Alondra Vega: Week 7	Alondra Vega: Week 12
Alondra Vega: Week 4	Alondra Vega: Week 8	Alondra Vega: Week 13
Alondra Vega: Week 5	Alondra Vega: Week 9	Alondra Vega: Week 14