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==Electronic Lab Notebook Week 10==
==Electronic Lab Notebook Week 10==
===Purpose===
===Purpose===
*The purpose of this assignment was to read a paper by Tai et al. (2007),outline the assigned paper, and prepare a journal club presentation to demonstrate our understanding of the content
===Part 1===
===Part 1===
=====Biological Terms=====
=====Biological Terms=====
Line 29: Line 31:
**The overall main result presented in this paper is that ''S. cerevisiae'' will have different transcriptional responses to low temperature and low specific growth rate when it is grown in batch cultures and in chemostat cultures.
**The overall main result presented in this paper is that ''S. cerevisiae'' will have different transcriptional responses to low temperature and low specific growth rate when it is grown in batch cultures and in chemostat cultures.
**The use of a chemostat cultures helps isolate and study the different physiological phases of ''S. cerevisiae'' and its ability to adapt to environmental stress.
**The use of a chemostat cultures helps isolate and study the different physiological phases of ''S. cerevisiae'' and its ability to adapt to environmental stress.
**
**ESR is not the main response to low temperatures.
**Transcriptional responses to low-temperature and low specific growth rate can be dissected by the use of chemostat cultures.
*'''What is the importance or significance of this work?'''
*'''What is the importance or significance of this work?'''
**The importance of this work is  
**The importance of this work is it demonstrates the benefits of using chemostat cultures versus batch cultures.
***Allows to see how the yeast cells react to changing environmental stresses in different physiological phases.
**There is more transcriptional programming in the low-temperature acclimation of yeast.
*'''Create a flow chart to describe their methods. Answer the following questions if they are relevant to your article.'''
*'''Create a flow chart to describe their methods. Answer the following questions if they are relevant to your article.'''
**Grow ''S. cerevisiae''in a chemostat --> grown at 12°C or grown at 30°C --> limit glucose or limit ammonia
**Grow ''S. cerevisiae''in a chemostat --> grown at 12°C or grown at 30°C --> limit glucose or limit ammonia
Line 56: Line 61:
**Glucose-limited and ammonium-limited feed were also used to eliminate the impact of secondary feeds.
**Glucose-limited and ammonium-limited feed were also used to eliminate the impact of secondary feeds.
*'''How many replicates did they perform per condition?'''
*'''How many replicates did they perform per condition?'''
**
**It appears that they performed three independent steady-state chemostat cultivations for each of the four different growth conditions.
*'''What mathematical/statistical method did they use to analyze the data?'''
*'''What mathematical/statistical method did they use to analyze the data?'''
**
**The cells were sampled from three chemostats, probes were prepared, and hybridized to Affymetrix Genechip microarrays.
**Agilent 2100 Bioanalyzer was used to determined RNA quality
**Microsoft Excel was used to to look for pair-wise comparisons by running a significance analysis of the microarray add-in.
**Expressionist Analyst version 3.2 was used to generate venn diagrams and heat-map visualizations of transcript data
**Regulatory Sequence Analysis (RSA) Tools were used to performed promoter analysis
**Database for Annotation, Visualization and Integrated Discovery (DAVID) 2006 were used for the statistical assessment of over-representation of GO biological processes categories among sets of significantly changed transcripts
**The Fisher's test is used to calculate the overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation (ChIP)-on-chip analysis
*'''What transcription factors did they talk about?'''
*'''What transcription factors did they talk about?'''
**
**Transciption factors mentioned and can be found specifically in table 2: Msn2/Msn4, Skf1p, Stt4p,Ino2/Ino4, Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-Dal82, Aft2p, Hsf1p, Nrg1p, Phd1p, Rcs1p, Rox1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, and Sok2-Nrg1.
*'''Table 1'''
*'''Table 1:''' Physiological characteristics of ''S. cerevisiae'' grown in ammonium- and glucose-limited anaerobic chemostat cultures
**
[[Image:CRTable1.png|thumb|right|Table 1]]
*'''Figure 1'''
**Values represent ±SD of data from three independent steady-state chemostat cultivations.
**
**Y glu/x is the biomass yield on glucose
*'''Figure 2'''
**DW is the dry weight
**
**GLU is glucose
*'''Table 2'''
**ETOH is ethanol
**
**CO2 is carbon dioxide
*'''Table 3'''
**The biomass yields and fermentation rates were similar at 12°C and 30°C in the glucose-limited and ammonium-limited chemostat cultures. This indicates that growth efficiency was not severely affected by the growth temperature.
**
*'''Figure 1''' Global transcriptome responses to anaerobic growth at 12°C and 30°C in anaerobic glucose- and ammonium-limited chemostat cultures.
*'''Figure 3'''
[[Image:CRFigure1.png|thumb|right|Figure 1]]
**
**There were 202 up regulated genes and 369 down regulated genes in the ammonium-limited media.
*'''Figure 4'''
**There were 123 up regulated genes and 136 down regulated genes in the glucose-limited media.
**
**There were 96 up regulated genes and 139 down regulated genes in the glucose-limited and ammonium-limited media.
*'''Figure 5'''
*'''Figure 2''' Heat map representing the transcript level ratio 1065 differently expressed genes in anaerobic glucose-limited and ammonium-limited chemostat cultures grown at 12°C and 30°C.
**
[[Image:CRFigure2.png|thumb|right|Figure 2]]
*'''Figure 6'''
**The genes indicated in the figure belong to the enriched GO categories.
**
**Nineteen NCR-responsive genes are underlined.
 
***Categories include: lipid metabolism, carbohydrate transport, rRNA processing, electron transport, amino acid transport, hexose metabolism, protein synthesis/protein complex assembly, ribosome biogenesis and assembly/RNA processing, nitrogen compound metabolism/amine transport/allantoin metabolism, polysaacharide and trehalose metabolism, M phase of mitotic cell cycle and chromosome segregation, cellular morphogenesis, response to stimulus, nuclear export, ribosome biogenesis and assembly, carbohydrate metabolism, response to stimulus, transport
*'''Table 2''' Protein and storage carbohydrates contents of ''S cerevisiae'' biomass grown in ammonium- and glucose-limited anaerobic chemostat cultures
[[Image:CRTable2.png|thumb|right|Table 2]]
**Values represent ±SD of data from three independent steady-state chemostat cultivations.
**Measurements include growth temperature, biomass dry weight, whole cell protein, biomass nitrogen content, trehalose, and glycogen.
**Trehalose and glycogen contents are significantly lower at 12°C than 30°C in ammonium-limited cultures.
**Trehalose contents were lower at 12°C, but the glycogen content was higher than it was at 30°C in glucose-limited cultures.
*'''Table 3''' Identification of (A) significantly overrepresented cis-regulatory binding motifs in 5' upstream regions and (B) significantly overrepresented promoter elements that bind known transcription factors (TF) or TF pairs according to ChiP-on-chip analysis in low temperature up- and down-regulated gene clusters derived from C-Lim and N-Lim chemostat experiments.
[[Image:CRTable3.png|thumb|right|Table 3]]
**(A) Significantly overrepresented ''cis''-regulatory binding motif in 5' upstream regions
**(B) Significantly overrepresented promoter elements that bind known transcription factors or transcription factor pairs according to ChiP-on-chip analysis
*'''Figure 3''' Genes differentially expressed in batch cultures during adaptation to low temperature. (A) Venn diagram showing the number of genes that are common to three batch-culture studies on low-temperature transcriptional adaptation. (B) Heat map representing the transcript ratio of 259 genes found in common in the three batch-culture low-temperature transcriptome datasets.
[[Image:CRFigure3.png|thumb|right|Figure 3]]
**Responses of 259 genes was not consistent, as few as 91 were consistently up-regulated at low temperatures, and only 48 were consistently down regulated at low temperatures.
*'''Figure 4''' Comparison of the transcript ratio of 259 genes common to three batch-culture low-temperature transcriptome datasets with the 12°C/30°C ratio of the genes specifically up- and down-regulated in anaerobic glucose- and ammonium-limited chemostat cultures. Venn diagrams show the number of low-temperature-responsive genes common to the batch-culture and chemostat-based datasets. The heat map shows the expression rations of the genes common to batch and chemostat datasets. The genes indicated between brackets show a consistent transcriptional regulation at low temperature in all datasets.
[[Image:CRFigure4.png|thumb|right|Figure 4]]
**Of the 29 genes that were transcriptionally regulated during both adaptation and acclimation to low temperature, only 11 genes showed a consistent pattern of regulation in all four situations.
**The four genes are: ''PIR3,'' ''SFK1,'' ''YPC1,''YEL073C,YNL024C, and YLR225C.
**''SFK1,'' ''YPC1,'' and YEL073C were consistently up-regulated at low temperature and are involved in lipid metabolism.
*'''Figure 5''' Comparison of the genes specifically up- or down-regulated during acclimation (this study) or adaptation (overlap of other datasets) to low temperature with the growth rate-dependent genes identified by Castrillo et al (2007) snf Regenberg et al (2006).
[[Image:CRFigure5.png|thumb|right|Figure 5]]
**There were a lot more genes that were up-regulated and down-regulated in this study compared to other growth rate studies and batch studies.
**The specific growth rate, rather than the temperature is primarily related to the altered transcript levels of the low-temperature down-regulated genes and altered levels of low-temperature up-regulated genes.
***This was seen in 25% of the low-temperature down-regulated genes and 10% of the low-temperature up-regulated genes.
*'''Figure 6''' Comparison of the genes specifically up- or down- regulated during acclimation (this study) or adaptation (overlap datasets from other studies) to low temperature with the ESR genes identified by Gasch et al. (2000).
[[Image:CRFigure6.png|thumb|right|Figure 6]]
**An extensive overlap was revealed when the set of ESR genes defined by Gasch et al. (2000) was compared with sets of genes that were consistently up-regulated or down-regulated at low temperatures in batch cultures.
***50% of the consistently low-temperature up-regulated genes and 13% of the low-temperature down-regulated genes were found in the batch culture studies and in the ESR genes.
**From part A of the figure, 1/3 of the low-temperature-responsive genes found in the three batch culture studies could be linked to ESR.
**There was an opposite transcriptional response in the low-temperature chemostat cultures for several genes that were up-regulated or down-regulated in the Gasch et al. (2000) study.
***This suggests that there is an alleviation of the environmental stress at low temperatures.
***Demonstrates that ESR is not an obligatory response to growth at low temperatures. ESR occurs during adaptation upon a sudden exposure to sub-optimal temperatures.
===Presentation===
*The journal club presentation can be found [[media:CRJournalClub-2.pptx|here]]
===Acknowledgments===
===Acknowledgments===
*I exchanged text messages with [[User: Lauren M. Kelly| Lauren M. Kelly]] on the night of Tuesday, March 28
*Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
*Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
*'''[[User:Cameron M. Rehmani Seraji|Cameron M. Rehmani Seraji]] 01:30, 30 March 2017 (EDT)'''


===References===
===References===

Latest revision as of 22:30, 29 March 2017

Electronic Lab Notebook Week 10

Purpose

  • The purpose of this assignment was to read a paper by Tai et al. (2007),outline the assigned paper, and prepare a journal club presentation to demonstrate our understanding of the content

Part 1

Biological Terms

Part 2

Outline

  • What is the main result presented in this paper?
    • The overall main result presented in this paper is that S. cerevisiae will have different transcriptional responses to low temperature and low specific growth rate when it is grown in batch cultures and in chemostat cultures.
    • The use of a chemostat cultures helps isolate and study the different physiological phases of S. cerevisiae and its ability to adapt to environmental stress.
    • ESR is not the main response to low temperatures.
    • Transcriptional responses to low-temperature and low specific growth rate can be dissected by the use of chemostat cultures.
  • What is the importance or significance of this work?
    • The importance of this work is it demonstrates the benefits of using chemostat cultures versus batch cultures.
      • Allows to see how the yeast cells react to changing environmental stresses in different physiological phases.
    • There is more transcriptional programming in the low-temperature acclimation of yeast.
  • Create a flow chart to describe their methods. Answer the following questions if they are relevant to your article.
    • Grow S. cerevisiaein a chemostat --> grown at 12°C or grown at 30°C --> limit glucose or limit ammonia
    • This results in there being four types of growth conditions:
      • 12°C, glucose-limited
      • 12°C, ammonium-limited
      • 30°C, glucose-limited
      • 30°C, ammonium-limited
  • How did they treat the cells (what experiment were they doing?)
    • The goal of the study was to investigate steady-state growth of S. cerevisiae at suboptimal temperatures, with emphasis on genome-wide transcriptional regulation.
  • What strain(s) of yeast did they use? Was the strain haploid or diploid?
    • The strain of yeast they used is prototrophic and haploid. The reference for the strain is S. cerevisiae strain CEN.PK113-7D (MATa)
  • What media did they grow them in? Under what conditions and temperatures?
    • Chemostat
      • They grew the yeast in a 2.0 L chemostat with a working volume of 1.0 L.
      • The dilution rate was set at 0.03 h^-1.
      • The pH was kept at 5.0 by automatic addition of 2 M KOH.
      • The stirrer speed was set at 600 rpm.
    • The yeast was grown at 12°C or 30°C.
    • The yeast was nitrogen-limited or glucose-limited.
    • Biomass dry weight, metabolites, dissolved oxygen, and gas profiles were constant for at least three volume changes before sampling occurred.
  • What controls did they use?
    • The yeast grown at 30°C was used as a control for the yeast grown at 12°C.
    • Glucose-limited and ammonium-limited feed were also used to eliminate the impact of secondary feeds.
  • How many replicates did they perform per condition?
    • It appears that they performed three independent steady-state chemostat cultivations for each of the four different growth conditions.
  • What mathematical/statistical method did they use to analyze the data?
    • The cells were sampled from three chemostats, probes were prepared, and hybridized to Affymetrix Genechip microarrays.
    • Agilent 2100 Bioanalyzer was used to determined RNA quality
    • Microsoft Excel was used to to look for pair-wise comparisons by running a significance analysis of the microarray add-in.
    • Expressionist Analyst version 3.2 was used to generate venn diagrams and heat-map visualizations of transcript data
    • Regulatory Sequence Analysis (RSA) Tools were used to performed promoter analysis
    • Database for Annotation, Visualization and Integrated Discovery (DAVID) 2006 were used for the statistical assessment of over-representation of GO biological processes categories among sets of significantly changed transcripts
    • The Fisher's test is used to calculate the overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation (ChIP)-on-chip analysis
  • What transcription factors did they talk about?
    • Transciption factors mentioned and can be found specifically in table 2: Msn2/Msn4, Skf1p, Stt4p,Ino2/Ino4, Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-Dal82, Aft2p, Hsf1p, Nrg1p, Phd1p, Rcs1p, Rox1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, and Sok2-Nrg1.
  • Table 1: Physiological characteristics of S. cerevisiae grown in ammonium- and glucose-limited anaerobic chemostat cultures
Table 1
    • Values represent ±SD of data from three independent steady-state chemostat cultivations.
    • Y glu/x is the biomass yield on glucose
    • DW is the dry weight
    • GLU is glucose
    • ETOH is ethanol
    • CO2 is carbon dioxide
    • The biomass yields and fermentation rates were similar at 12°C and 30°C in the glucose-limited and ammonium-limited chemostat cultures. This indicates that growth efficiency was not severely affected by the growth temperature.
  • Figure 1 Global transcriptome responses to anaerobic growth at 12°C and 30°C in anaerobic glucose- and ammonium-limited chemostat cultures.
Figure 1
    • There were 202 up regulated genes and 369 down regulated genes in the ammonium-limited media.
    • There were 123 up regulated genes and 136 down regulated genes in the glucose-limited media.
    • There were 96 up regulated genes and 139 down regulated genes in the glucose-limited and ammonium-limited media.
  • Figure 2 Heat map representing the transcript level ratio 1065 differently expressed genes in anaerobic glucose-limited and ammonium-limited chemostat cultures grown at 12°C and 30°C.
Figure 2
    • The genes indicated in the figure belong to the enriched GO categories.
    • Nineteen NCR-responsive genes are underlined.
      • Categories include: lipid metabolism, carbohydrate transport, rRNA processing, electron transport, amino acid transport, hexose metabolism, protein synthesis/protein complex assembly, ribosome biogenesis and assembly/RNA processing, nitrogen compound metabolism/amine transport/allantoin metabolism, polysaacharide and trehalose metabolism, M phase of mitotic cell cycle and chromosome segregation, cellular morphogenesis, response to stimulus, nuclear export, ribosome biogenesis and assembly, carbohydrate metabolism, response to stimulus, transport
  • Table 2 Protein and storage carbohydrates contents of S cerevisiae biomass grown in ammonium- and glucose-limited anaerobic chemostat cultures
Table 2
    • Values represent ±SD of data from three independent steady-state chemostat cultivations.
    • Measurements include growth temperature, biomass dry weight, whole cell protein, biomass nitrogen content, trehalose, and glycogen.
    • Trehalose and glycogen contents are significantly lower at 12°C than 30°C in ammonium-limited cultures.
    • Trehalose contents were lower at 12°C, but the glycogen content was higher than it was at 30°C in glucose-limited cultures.
  • Table 3 Identification of (A) significantly overrepresented cis-regulatory binding motifs in 5' upstream regions and (B) significantly overrepresented promoter elements that bind known transcription factors (TF) or TF pairs according to ChiP-on-chip analysis in low temperature up- and down-regulated gene clusters derived from C-Lim and N-Lim chemostat experiments.
Table 3
    • (A) Significantly overrepresented cis-regulatory binding motif in 5' upstream regions
    • (B) Significantly overrepresented promoter elements that bind known transcription factors or transcription factor pairs according to ChiP-on-chip analysis
  • Figure 3 Genes differentially expressed in batch cultures during adaptation to low temperature. (A) Venn diagram showing the number of genes that are common to three batch-culture studies on low-temperature transcriptional adaptation. (B) Heat map representing the transcript ratio of 259 genes found in common in the three batch-culture low-temperature transcriptome datasets.
Figure 3
    • Responses of 259 genes was not consistent, as few as 91 were consistently up-regulated at low temperatures, and only 48 were consistently down regulated at low temperatures.
  • Figure 4 Comparison of the transcript ratio of 259 genes common to three batch-culture low-temperature transcriptome datasets with the 12°C/30°C ratio of the genes specifically up- and down-regulated in anaerobic glucose- and ammonium-limited chemostat cultures. Venn diagrams show the number of low-temperature-responsive genes common to the batch-culture and chemostat-based datasets. The heat map shows the expression rations of the genes common to batch and chemostat datasets. The genes indicated between brackets show a consistent transcriptional regulation at low temperature in all datasets.
Figure 4
    • Of the 29 genes that were transcriptionally regulated during both adaptation and acclimation to low temperature, only 11 genes showed a consistent pattern of regulation in all four situations.
    • The four genes are: PIR3, SFK1, YPC1,YEL073C,YNL024C, and YLR225C.
    • SFK1, YPC1, and YEL073C were consistently up-regulated at low temperature and are involved in lipid metabolism.
  • Figure 5 Comparison of the genes specifically up- or down-regulated during acclimation (this study) or adaptation (overlap of other datasets) to low temperature with the growth rate-dependent genes identified by Castrillo et al (2007) snf Regenberg et al (2006).
Figure 5
    • There were a lot more genes that were up-regulated and down-regulated in this study compared to other growth rate studies and batch studies.
    • The specific growth rate, rather than the temperature is primarily related to the altered transcript levels of the low-temperature down-regulated genes and altered levels of low-temperature up-regulated genes.
      • This was seen in 25% of the low-temperature down-regulated genes and 10% of the low-temperature up-regulated genes.
  • Figure 6 Comparison of the genes specifically up- or down- regulated during acclimation (this study) or adaptation (overlap datasets from other studies) to low temperature with the ESR genes identified by Gasch et al. (2000).
Figure 6
    • An extensive overlap was revealed when the set of ESR genes defined by Gasch et al. (2000) was compared with sets of genes that were consistently up-regulated or down-regulated at low temperatures in batch cultures.
      • 50% of the consistently low-temperature up-regulated genes and 13% of the low-temperature down-regulated genes were found in the batch culture studies and in the ESR genes.
    • From part A of the figure, 1/3 of the low-temperature-responsive genes found in the three batch culture studies could be linked to ESR.
    • There was an opposite transcriptional response in the low-temperature chemostat cultures for several genes that were up-regulated or down-regulated in the Gasch et al. (2000) study.
      • This suggests that there is an alleviation of the environmental stress at low temperatures.
      • Demonstrates that ESR is not an obligatory response to growth at low temperatures. ESR occurs during adaptation upon a sudden exposure to sub-optimal temperatures.

Presentation

  • The journal club presentation can be found here

Acknowledgments

  • I exchanged text messages with Lauren M. Kelly on the night of Tuesday, March 28
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
  • Cameron M. Rehmani Seraji 01:30, 30 March 2017 (EDT)

References

  • Week 10 Assignment Page
  • Tai, S. L., Daran-Lapujade, P., Walsh, M. C., Pronk, J. T., & Daran, J. M. (2007). Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis. Molecular biology of the cell, 18(12), 5100-5112. doi: 10.1091/mbc.E07-02-0131

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