Carmen E. Castaneda: Week 9

Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. You can use the glossary in any molecular biology, cell biology, or genetics text book as a source for definitions, or you can use one of many available online biological dictionaries (links below). List the citation(s) for the dictionary(s) you use, providing a URL to the page is fine.

1. trehalose is a naturalalpha linked disaccharide (1)
2. osmolarity is the measure of solute concentration (2)
3. dendrogram is a type of graph which demonstrates clustering(3)
4. ribosome biogenesis the process incharge of making ribosomes (4)
5. glycogen is a type of polymer of glucose (5)
6. diauxic has to do with the french word having to do with two growth phase (6)
7. Bactopeptone is a type of enzymatic digest rich in amino acids (7)

1. Write an outline of the article. The length should be the equivalent of 2 pages of standard 8 1/2 by 11 inch paper (you can check this by clicking on "Print preview" in your browser). Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.
   • What is the main result presented in this paper?
   • What is the importance or significance of this work?
   • How did they treat the cells (what experiment were they doing?)
   • What strain(s) of yeast did they use? Was the strain haploid or diploid?
   • What media did they grow them in? What temperature? What type of incubator? For how long?
   • What controls did they use?
   • How many replicates did they perform per timepoint?
   • What mathematical/statistical method did they use to analyze the data?
   • What transcription factors did they talk about?
   • Briefly state the result shown in each of the figures and tables.

Outline

Abstract

• transcriptional response of budding yeast to cold
  • exposed to 10°C for different lengths of time
  • DNA microarrays were used characterize the changes in transcript abundance
    • two main groups were identified, early cold response, ERC, and late cold response, LRC
  • overlap between environmental stress response, ESR, genes and LRC
  • acoomulation of the carbohydrate reserve trehalose and glycogen is indunced during LRC
  • strand munipulation
    • Msn2p and Msn4p involvement with the inductionof genes common to stress responses
      • recognition of paterns during LRC and comparirison between ERC

Introduction

• Unicellular organisms are affected by a variety of extreme changes in their environments
  • Changes in their environment like nutrients, acidity, osmolarity temperature, toxins and radiation
• Developed programmed responses to stress into the genetic code, not random
  • The transcription of genes is changed
    • which is general stress response.
• ~10% of the genome responds
  • Genes involved are defined as environmental stress response, ESR
    • Induced ESR genes tend to be those involved in a variety of cell functions like protein folding &degradation, transport, & carbohydrate metabolism
    • Repressed deal with cell growth
• Little known about mechanisms responsible for growth and survival at low temperatures
• Cold causes different changes in the physical and biochemical properties
• Ability to adapt is determined by different regulatory mechanisms
• We'll compare cold responses to the responses of other stress stimuli and accumulation of trehalose and glycogen in cold stress

Materials and Methods

• Strains
  • BY4743 (wild type)
  • BSY25
    • derived from a cross of the two single-mutant strains
  • W303
    • for growth curve experiments
• growth medium and culture condition
  • YPD medium - 2% glucose, 2% bactopeptone, 1% yeast extract
  • culture inoculated from fresh colony and grown overnight at 30°C in 50 ml of medium in Erlenmeyer flasks shaken at 170rpm
    • these were the diluted to 0.05 OD in 500ml of fresh medium, grown to 0.6 OD at 30°C in 1500ml flasks shaken 170rpm
      • transferred to a 10°C water bath shaker, in which they were incubated for 10, 30 or 120 min at 170rpm before being harvested
  • temperature dropped 4°C per minute
• isolation of RNA
  • isolated using hot-phenol method with modifications
    • 500ml cells were processed in 50 ml tubes by extracting phenol
• 3 microarrays of mRNA were labled directly using Cy3- and Cy5-CTP through reverse transcription
• microarray slides were scanned using ScanArray lite scanner
  • normalization and quality controls of the files were performed in Excel
  • Log2 values of the ratio for each duplicate spot were averaged
• time-course experiments with the wild-type strain were performed with time points of 0,2,12 hr(repeated twice)
  • • and at 10min, 30min, 60 hr (three independent repeats)
  • two repeats on thesinle mutant strains
  • 43 microarrays were used

Results

• cold response of S. cerevisiae
  • subjected to low temperature (10°C), demostrated a reduction growth rate but a normal growth curve
  • yeast cells respond to a rapid temperature shift from 30-10°C with transient changes in gene expression
  • figure 1A:data was collected and organized in two dimensional hierarchical clustering.
    • Along the horizontal we have genes clustered
    • On the vertical axis the individual chips are clustered
    • LCR genes are more active
    • As the temperature begins to drop we have more genes activated and responding to the change in temperature
    • a peak in activity at the 12 hr mark were 2/3 of the genes are either down regulated, green, or up, red
    • D-F are considered the ECR genes while, LCR are A-C.
  • Figure 1B,C: show different classes of genes in both the ECR and LCR and whether they were up or down regulated
    • we see there are more genes being stimulated during the LCR
• ECR
  • genes being reproducibly induced ≥ 2fold
  • rapidly decreases the levels of expression of some genes
  • 32 genes were reduced within the first 2 hrs
• LCR
  • 280 genes were reproducibly induced twofold or more at 12/60 hr
• 2h period time point to represent ECR and the 12 h time point to represent LCR since max cahnges were recorded at this time
• figure 2: a comparison of the ECR with transcriptional pattern produced by decrease in temperature showed a similar transcriptional response with time dependency
  • compared gasch result with the experiment
    • had a inverse result
• majority of repressed ECR genes were repressed during temperature decrease
• figure 3A:compares ECR expression with different stress stimuli
• figure 3B:compares LCR expression with different stress stimuli
• figure 3C top: compare and contrast the number of genes induced or repressed accoriding to the stress genes
  • 87 genes were induced between LCR and ESR
  • 111 genes were repressed between LCR and ESR
• figure 3C bottom: compare and contrast the number of genes induced or repressed accoriding to the genes
  • 2 genes were induced between ECR and ESR
  • 2 genes were repressed between ECR and ESR
    • The ESR genes do not have such a strong relationship with ERC as they do with LCR
• the Δmsn2 Δmsn4 strain was characterized during growth at different steady states temperatures
  • cold induced genes dependent of these genes show no activitation in mutant strain
  • decrease in intensity relative to wild-type strain
  • cold repressed Msn2p/Msn4p-dependent genes in crease in relative signal intensity
• figure 4:relative expression of 120 genes were affected ≥2fold in the Δmsn2 Δmsn4 strain exposed to cold
• Msn2p/Msn4p were required for activation of 99 LCR genes
• 78% of the LCR genes were unaffected by the absence of Msn2p/Msn4p
  • LCR gene expression is involved with these genes
• an accumalation of carbohydrates, glycogen and trehalose
• figure 5:we see the accumalation of gyclogen and trehalose over the course of the time
  • the accumulation incresed over time
    • more glycogen present than trehalose at 60 h

Discussion

• the study modeled S. cerevisiae to study the transcriptional response to abrupt changes in temperature
• the yeast initiates different expression programs during th response to cold
• two groups were identified,ECR and LCR
• figure 6:compares the gasch global stress-transcription profiles with the gene expression of this study
  • during ECR, major differences are apparent