Nick Rohacz: Week 9

Vocab

 * 1) Shine-Dalgarno sequence - A short sequence of nucleotides on a prokaryotic mRNA molecule, that serves to bind to ribosomal RNA and bring the ribosome to the start codon on the mRNA.
 * 2) Dimorphic Fungi - two different forms of Fungi.
 * 3) Ubiquitin - small regulatory protein that is found almost anywhere in Eukaryotes.
 * 4) Hybridization - The process of forming a double stranded nucleic acid from joining two complementary strands of DNA (or RNA).
 * 5) Photomultiplier tube - light detectors that are useful in low intensity applications.
 * 6) Diauxic shift - A growth phase followed by a lag phase, after which growth is resumed.
 * 7) Hyperosmotic - characterized by having increased osmotic pressure.
 * 8) Superoxide-generating drug - a drug that generates O-2 ions
 * 9) Desaturase - Any enzyme that puts double bonds in the hydrocarbon tail portion of fatty acids.
 * 10) Sonicated - The process of dispersing, disrupting, or inactivating biological material by sound waves.

Outline

 * What is the main result presented in this paper?
 * 1) The transcriptional responses of budding yest S. cerevisiae to early cold response (ECR) and late cold response (LCR) were determined. The transcriptional response to LCR was similar to that of environmental stress response (ESR) whereas the ECR was seemingly unique.
 * What is the importance or significance of this work?
 * 1) The significance of this work is to see how Eukaryotic cells respond to Cold Stress.
 * How did they treat the cells (what experiment were they doing?)
 * 1) Initially grown at 30C and then cells kept in early log phase at 10C for different time intervals. Perform microarray testing on yeast RNA.
 * What strain(s) of yeast did they use? Was the strain haploid or diploid?
 * 1) BY4743(wild type, haploid) and BSY25(BY4743 except homozygous, haploid)
 * What media did they grow them in? What temperature? What type of incubator? For how long?
 * Yeast Extract Peptone Dextrose medium (YPD): 2% glucose, 2% bactopeptone, 1% yeast extract . Grown at 30 degrees dropped to 10 degrees at 4 C/min. Grown in 50 ml of medium in 250 ml Erlenmeyer flask shaken at 170 RPM
 * What controls did they use?
 * 1) Cys dyes were swapper out between control and experimental samples
 * 2) Control was kept at 30C and all data points were compared to similar data.
 * How many replicates did they perform per timepoint?
 * 1) 2 replicates for 0, 2, and 12 h. 3 replicates for 10 min, 30 min, and 60 hour. 2 for each with mutant except 3 for 12h
 * What mathematical/statistical method did they use to analyze the data?
 * 1) Hierarchical clustering by GeneSpring software using standard correlation.
 * What transcription factors did they talk about?
 * 1) Msn2p, Msn4p; these bind to stress response elements
 * 2) Spt23p, Mga2p: ER membrane bound transcription factors that regulate OLE1 (OLE1 is a gene involved in lipid metabolism and is responsible for membrane fluidity.
 * 3) HAP5 and TYE7: carbohydrate metabolism transcription factors induced in LCR
 * Briefly state the result shown in each of the figures and tables.

Introduction

 * Molecular basis of the response to many different stresses have been studied in S. cerevisiae.
 * Yeast cells undergoing heat shock induce heat shock proteins regulated by the transcription factor Hsf1p
 * Induced environmental stress response (ESR’s) genes are required for a variety of cellular functions(1), while repressed ESR genes usually function in cell growth(2).
 * 1) ex. Protein folding and degradation, transport, and carbohydrate metabolism.
 * 2) ex. RNA metabolism, nucleotide biosynthesis, secretion, and ribosomal performance.
 * Approximately 10% of the genome is induced or repressed in the presence of stress, regulated by the two transcription factors Msn2p and Msn4p.
 * Want to test cold stress, known physical change during cold stress is decrease in membrane fluidity. Occurs by adding double bonds to hydrocarbon tail of fatty acid with desaturase.

Strains

 * Used BY4743(wild type) and BSY25(BY4743 except homozygous), for growth curve experiment, W303(wild type) was also used.

Growth Medium and Culture Conditions

 * Cultures grown in YPD medium, cells grown overnight at 30°C and shaken at 170 rpm, batches were then diluted and harvested by centrifugation at 10 or 30°C for 2min at 3500 rpm.. Final dilution of 0.6-0.8 OD600 before snap frozen and stored.

Isolation of RNA

 * Total RNA isolated using hot-phenol method, for 60hr, RNA extraction improved by adding glass beads.

RNA labeling and DNA microarray hybridization

 * RNA was labeled with Cy3- or Cy5- dye through reverse transcription. Prehybridization done in 20:1:1 of DigEasyHyb solution: yeast tRNA: sonicated salmon sperm DNA.
 * Microarrays washed with buffer, airstream dried and immediately hybridized.

Data acquisition and Analysis

 * To be included in normalization and analysis, each DNA had to pass three quality controls:
 * 1) signal intensity must be significantly greater than local background.
 * 2) signal intensity must be within dynamic range of photomultiplier tube.
 * 3) raw intensities of duplicate spots must be within 50% of each other.
 * To correct for variation in local intensities, performed a subarray normalization on each individual subarray.

Experimental Design

 * To ensure same physiological state, sampled index of cells and glucose content, found to be 70% and ~16g/l on average.
 * To ensure no diauxic shift occurred during continuous growth, diauxic shift-inducible genes were analyzed, no transcriptional change occurred.
 * Time points are 0hr, 10min, 30min, 2hr, 12hr, and 60hr.
 * One wild type and two Δmsn2Δmsn4 strain time course experiments were run, except 12hr, three strains were run, control microarrays were run, independent strains as 30C, three repeats.

Comparison with other S. cerevisiae Stress Data

 * Compared ESR genes, responses to stresses, and the expression data for cold response with multiple other souces.

Biochemical and Analytical procedure

 * Used other sources for determination of glycogen and trehalose procedures. Glucose concentrations determined using the Glucose kit.

Results

 * Figure 1: Overall look at the transcriptional response to cold for wild-type experiment. A: cluster analysis of microarray data for 634 genes that were determined to be significant. Green: repressed, red: induced. Early Cold Response genes shown in sections labeled D and E. A, B, and C show Late Cold Response genes. Classification and amount of such genes for ECR in B and LCR in C.


 * Figure 2: Comparison of data of ECR from 30 to 10 degrees is found in the shift from 37 deg to 25 deg C as in the Gasch experiment. Only the genes labeled a and b on the diagram were found to be in correlation with control.


 * Figure 3: ECR compared with LCR, both individually compared to ESR and the genes used in both are compared in Venn Diagrams with the Environmental Stress Response. It is found that the ECR only shares 2 induced genes and 2 repressed genes with the ESR whereas the LCR and the ESR share 87 induced genes and 111 repressed genes. Responses to various drugs are also investigated.


 * Figure 4: Comparison of wild type gene expression for 2 hour and 12 hour time points vs the delta msn2 delta msn4 strains. This data suggests that the ECR expression profile is cold-specific whereas the LCR genes are similar to that of ESR.


 * Figure 5: Investigation of amounts of glycogen and trehalose. The mutant strain did not produce a significant amount of trehalose at any time point and produced less glycogen than the wild type at all time points. The mutant strain was less able to accumulate reserve carbohydrates during cold treatment.


 * Figure 6: Comparison of transcriptional response in wild type with the Sahara (2002) study. A common cluster of genes during the LCR was observed, this list includes several general stress-response genes. The data for the ECR varied drastically, however between the two data sets. 2002 Sahara paper indicated ribosomal genes would be induced, while Schade paper noticed decrease in transcript abundance. This could be explained by difference in yeast strain or in the phase

Closing

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

Nicholas A. Rohacz 03:03, 24 March 2011 (EDT)