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==List of 10 Unknown Words and Their Definitions==
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=Article=
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#
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Sahara, T., Goda, T., & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277(51), 50015-50021.
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==Outline of Article==
+
=List of 10 Unknown Words and Their Definitions=
−
I. Introduction
+
# hypoxia - low oxygen levels in the blood (Ratini, 2016)
−
*
+
# ubiquitin - plays a role in the heat-shock response, is involved in quality control of nascent proteins, membrane trafficking, cell signalling, cell cycle control, X chromosome inactivation and the maintenance of chromosome structure (Lackie, 2013)
−
*
+
# Cytosolic - contents of the fluid in the cytoplasm of a cell (King, et al., 2014).
−
*
+
# diauxic shift - the two-phase growth response seen in a culture of microorganisms making a phenotypic adaptation to the addition of a second substrate (AccessScience, 2015)
−
*
+
# methyl methanesulfonate - A DNA damaging agent to induce mutagenesis and in recombination experiments (Lundin, 2005).
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II. Methods
+
# peptidyl-prolyl cis/trans- isomerases - catalyse the cis–trans isomerisation of peptide bonds N-terminal to proline residues in polypeptide chains, play a role in the folding of newly synthesised proteins, and assist in cell cycle control (Shaw, 2002)
−
*
+
# trehalose - a sugar thought to be implicated in anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation (National Center for Biotechnology Information, 2016)
−
*
+
# ''de novo'' - New; not present previously; just beginning (Honee, 2009)
−
*
+
# midlgarithmic - cell numbers increase in a logarithmic fashion but have not reached their full reproduction rate (Rogers & Kadner, 2017)
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* FLOW CHART
+
# 2-fold - method of reporting statistics using log2 that results in more reproducible gene lists than do the ordinary and modified t-statistics (Witten & Tibshirani, 2007)
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III. Results and Discussion
 
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*
 
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*
 
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*
 
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IV. Conclusion
 
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*
 
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*
 
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*
 
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V. Further Implications
 
  
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==Deliverable==
+
= Flow Chart of Overall Experimental Design = 
 +
 
 +
[[File: Flowchart_Design.png]]
 +
 
 +
=Outline of Article=
 +
== Background Information on Purpose of Experiment ==
 +
* Low temperatures are known to have several effects on biochemical and physiological properties in various cells
 +
* Cold shock proteins are induced when cells are exposed to low temperatures in order to cope with the drastic change in environment 
 +
* In yeast, the NSR1, TIP1, and OLE1 genes have been identified as important cold-inducible genes through past research
 +
*low temperature-dependent gene expression and low temperature response are still unclear
 +
* '''The purpose of this experiment is to analyze global gene expression in low temperature-exposed yeast cells using a yeast cDNA microarray to obtain fundamental information on low temperature response and low temperature-dependent gene expression in yeast cells'''
 +
== Cold shock and microarray procedures of yeast samples ==
 +
* ''S. cerevisiae'' YPH500 was used for all the analyses
 +
* Cultured aerobically in YPD medium (yeast extract, peptone, and glucose) at 30°C and shaken at 100 rpm
 +
* 50 ml of the culture were collected for a reference time of 0
 +
* Cells flash-frozen in liquid nitrogen
 +
* Stored at -80°C in preparation for RNA
 +
* The remaining cells were cold shocked at 10°C then cultured at the same temperature
 +
* Cells collected at 0.25, 0.5, 2, 4, and 8 hours after the cold shock
 +
* Cells flash-frozen in liquid nitrogen
 +
* Stored at -80°C in preparation for RNA
 +
* Cy3-dUTP and Cy5-dUTP were used as cDNA probes
 +
* Labeled with fluorophore in order to carry out microarray hybridization
 +
* Microarray hybridization performed based on the manual for S. cerevisiae cDNA microarray
 +
* Microarrays were scanned by laser microscope
 +
* Process repeated twice
 +
* Averaged the expression ratios of the separate experiments for final data
 +
* Images analyzed by computer program
 +
* Data analyzed by analysis software
 +
* Fluorescence intensities were normalized
 +
* Data was clustered and referred to the Munich Information Center for Protein Sequences functional database
 +
* Functional relationships among the genes in each cluster was determined
 +
 
 +
== Results and Discussion ==
 +
=== Global Expression Analysis of Low Temperature Response in Yeast Cells Using a cDNA Microarray ===
 +
* Changes in expression of genes in yeast after cold shock was analyzed using cDNA of 5,803 genes in a yeast genome
 +
* Ratio of fluorescent intensities was 2 fold for a mainly all the cDNA spots on the data
 +
* Roughly 25% of yeast genes' expression levels were affected by cold shock
 +
* Increase in number of genes up-regulated
 +
* Increase in number of genes down-regulated
 +
* Significantly up or down-regulated genes were classified according to the MIPS functional database
 +
* Up-regulated and down-regulated genes increased in almost all categories when exposed to low temperatures
 +
** Suggests that changes in gene expression are due to the introduction of low temperatures in order to adapt to their environment
 +
** Other cells have demonstrated this ^^ when exposed to other environmental stresses
 +
=== Clustering Analysis of Global Expression Data ===
 +
* Genes close together were placed into clusters
 +
* Clusters showed:
 +
**Similar functions
 +
**Cooperative regulation
 +
*Clusters:
 +
**1A: Unclassified proteins
 +
**1B: Amino acid biosynthesis and metabolism
 +
**1C: RNA polym. I & RNA processing
 +
**1D: Ribosomal proteins
 +
**1E: Not defined
 +
* Up regulated genes were divided into three clusters depending on their expression profiles
 +
** IC: up-regulated after within 30 mins after cold shock
 +
** ID: high up-regulation at 2hr and at 4-8 hr
 +
** IE: high up-regulation at 2hr and at 4-8 hr
 +
=== Genes related to transcription ===
 +
* Further classification of genes:
 +
** 2A: RNA polymerase I & RNA processing - '''up regulated, then down regulated in later phase'''
 +
** 2B: rRNA processing - '''up regulated, then down regulated in later phase'''
 +
** 2C mRNA transcription - '''up-regulated in mid phase'''
 +
** 2D: mRNA transcription - '''continuously down regulated'''
 +
* These findings suggest that...
 +
** The mechanisms for transcription are up regulated when exposed to low temperatures in the early phase
 +
*** Up-regulated mRNAs = essential for basic transcriptional/translational functions, like encoding regulatory proteins for amino acid production
 +
** The genes for transcriptional regulation and mRNA synthesis made diverse responses in the late phase
 +
*** Down-regulated mRNAs = not essential for survival in cold shock, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes
 +
=== Ribosomal Protein Genes ===
 +
* Cluster Classification:
 +
** 3A & 3B: cytosolic ribosome - '''up regulated first, then down regulated in later phase'''
 +
** 3C: translational control factors - '''continuously up regulated'''
 +
** 3D: tRNA synthetases - '''continuously down regulated'''
 +
* These findings suggest that...
 +
** Low temperature impairs translational ability
 +
** Yeast genes up-regulate to compensate
 +
=== Cell Rescue, Defense, Death, and Aging ===
 +
* Cluster Classification:
 +
** 4A: not labeled
 +
** 4B & 4C: stress response - '''high up-regulation in mid-late phase'''
 +
** 4D: stress response and chaperone - '''high down-regulation in mid-late phase'''
 +
*These findings suggest that...
 +
** Heat shock protein genes down-regulated (EXCEPT for HSP12 and HSP26)
 +
** Protein folding genes up-regulated
 +
=== Metabolism and Energy Production ===
 +
* Cluster Classification:
 +
** 5A: nucleotide metabolism - '''up-regulated early-mid phase, then down-regulated'''
 +
** 5B & 5E: not specified
 +
** 5C & 5D: C-compound and carbohydrate metabolism - '''continuously up-regulated'''
 +
** 5F & 5H: amino acid metabolism - '''continuously down-regulated'''
 +
** 5G: C-compound and carbohydrate utilization - '''continuously down-regulated'''
 +
* These findings suggest that...
 +
** Glycogen and trehalose production genes showed a lot of cooperative up-regulation
 +
** Trehalose may help protect cellular membrane, which may help to keep yeast cells intact at low temperatures
 +
=== Signal Transduction Components ===
 +
* Cluster Classification:
 +
** 6A: signal transduction - '''down-regulated'''
 +
** 6B: signal transduction - '''up regulated in early phase, then down-regulation'''
 +
** 6C: signal transduction - '''down-regulated in early phase, then up-regulation in late'''
 +
* These findings suggest that...
 +
** Genes related to cAMP-PKA pathway and Msn2p/4p were up-regulated
 +
** Increase in PKA signaling has been known to be response to stresses
 +
** Increase in...
 +
*** Signaling
 +
*** Metabolism control
 +
*** Stress resistance
 +
== Conclusion ==
 +
* Gene expression changed to maintain transcription and translation and to adapt a tolerance to the colder temperature
 +
* Transcriptional genes up-regulated first to help transcription and translation
 +
* Ribosomal proteins up-regulated in the middle phase to further assist maintenance of translation
 +
* Stress response induced genes up-regulated in the late phase
 +
* Maintaining translation is priority to yeast in cold shock
 +
* Cells need to make proteins to help maintain integrity and basic functions of cells
 +
* Yeast cells can adapt to environment once ^^^ obtained to gain tolerance to low temperature
 +
* Other organisms show similar responses in gene expression when exposed to environmental stresses
 +
 
 +
=Deliverable=
 
[[Media:Cold_Shock_Yeast_Genome_Response.pdf | Journal Club Week 12 Presentation]]
 
[[Media:Cold_Shock_Yeast_Genome_Response.pdf | Journal Club Week 12 Presentation]]
  
−
==Acknowledgements==  
+
=Acknowledgements=
 +
I met with Corrine outside of class to discuss the method of presenting for the journal club. We worked on our presentation together and equally prepared for the assignment.
 +
While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. <br>
 +
[[User:Dbashour|Dbashour]] ([[User talk:Dbashour|talk]]) 11:57, 21 November 2017 (PST)
 +
 
 +
=References=
 +
*AccessScience. (2015). Diauxic growth (diauxie). Retrieved November, 2017, from https://www.accessscience.com/content/diauxic-growth-diauxie/BR0105151 <br>
 +
*Honee, V. (2009). De novo. Retrieved November, 2017, from http://www.biology-online.org/dictionary/De_novo <br>
 +
*King, R.C., Mulligan, P.K., & Stansfield, W.D. (Eds.). (2014). A Dictionary of Genetics. In Oxford Reference. Retrieved from http://www.oxfordreference.com/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444<br>
 +
*Lackie, J. M. (2013). The dictionary of cell and molecular biology. Oxford: Academic Press. <br>
 +
*LMU BioDB 2017. (2017). Week 12. Retrieved November 14, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12
 +
*Lundin, C. (2005). Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Research, 33(12), 3799-3811. doi:10.1093/nar/gki681 <br>
 +
* National Center for Biotechnology Information (2016). Trehalose. Retrieved November 2017, from https://pubchem.ncbi.nlm.nih.gov/compound/trehalose#section=Top <br>
 +
*Ratini, M. (2016). Hypoxia and Hypoxemia. Retrieved November, 2017, from https://www.webmd.com/asthma/guide/hypoxia-hypoxemia#1 <br>
 +
*Rogers, K., & Kadner, R. J. (2017). Growth of bacterial populations. Retrieved November, 2017, from https://www.britannica.com/science/bacteria/Growth-of-bacterial-populations#ref955458 <br>
 +
*Sahara, T., Goda, T., & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277(51), 50015-50021.
 +
*Shaw, P. E. (2002). Peptidyl‐prolyl isomerases: A new twist to transcription. EMBO reports, 3(6), 521-526. <br>
 +
*Witten, D., & Tibshirani, R. (2007). A comparison of fold-change and the t-statistic for microarray data analysis. Analysis, 1776, 58-85. <br>
  
−
==References==
+
{{template:dbashour}}

Latest revision as of 22:13, 21 November 2017

Article

Sahara, T., Goda, T., & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277(51), 50015-50021.

List of 10 Unknown Words and Their Definitions

  1. hypoxia - low oxygen levels in the blood (Ratini, 2016)
  2. ubiquitin - plays a role in the heat-shock response, is involved in quality control of nascent proteins, membrane trafficking, cell signalling, cell cycle control, X chromosome inactivation and the maintenance of chromosome structure (Lackie, 2013)
  3. Cytosolic - contents of the fluid in the cytoplasm of a cell (King, et al., 2014).
  4. diauxic shift - the two-phase growth response seen in a culture of microorganisms making a phenotypic adaptation to the addition of a second substrate (AccessScience, 2015)
  5. methyl methanesulfonate - A DNA damaging agent to induce mutagenesis and in recombination experiments (Lundin, 2005).
  6. peptidyl-prolyl cis/trans- isomerases - catalyse the cis–trans isomerisation of peptide bonds N-terminal to proline residues in polypeptide chains, play a role in the folding of newly synthesised proteins, and assist in cell cycle control (Shaw, 2002)
  7. trehalose - a sugar thought to be implicated in anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation (National Center for Biotechnology Information, 2016)
  8. de novo - New; not present previously; just beginning (Honee, 2009)
  9. midlgarithmic - cell numbers increase in a logarithmic fashion but have not reached their full reproduction rate (Rogers & Kadner, 2017)
  10. 2-fold - method of reporting statistics using log2 that results in more reproducible gene lists than do the ordinary and modified t-statistics (Witten & Tibshirani, 2007)

Flow Chart of Overall Experimental Design

Flowchart Design.png

Outline of Article

Background Information on Purpose of Experiment

  • Low temperatures are known to have several effects on biochemical and physiological properties in various cells
  • Cold shock proteins are induced when cells are exposed to low temperatures in order to cope with the drastic change in environment
  • In yeast, the NSR1, TIP1, and OLE1 genes have been identified as important cold-inducible genes through past research
  • low temperature-dependent gene expression and low temperature response are still unclear
  • The purpose of this experiment is to analyze global gene expression in low temperature-exposed yeast cells using a yeast cDNA microarray to obtain fundamental information on low temperature response and low temperature-dependent gene expression in yeast cells

Cold shock and microarray procedures of yeast samples

  • S. cerevisiae YPH500 was used for all the analyses
  • Cultured aerobically in YPD medium (yeast extract, peptone, and glucose) at 30°C and shaken at 100 rpm
  • 50 ml of the culture were collected for a reference time of 0
  • Cells flash-frozen in liquid nitrogen
  • Stored at -80°C in preparation for RNA
  • The remaining cells were cold shocked at 10°C then cultured at the same temperature
  • Cells collected at 0.25, 0.5, 2, 4, and 8 hours after the cold shock
  • Cells flash-frozen in liquid nitrogen
  • Stored at -80°C in preparation for RNA
  • Cy3-dUTP and Cy5-dUTP were used as cDNA probes
  • Labeled with fluorophore in order to carry out microarray hybridization
  • Microarray hybridization performed based on the manual for S. cerevisiae cDNA microarray
  • Microarrays were scanned by laser microscope
  • Process repeated twice
  • Averaged the expression ratios of the separate experiments for final data
  • Images analyzed by computer program
  • Data analyzed by analysis software
  • Fluorescence intensities were normalized
  • Data was clustered and referred to the Munich Information Center for Protein Sequences functional database
  • Functional relationships among the genes in each cluster was determined

Results and Discussion

Global Expression Analysis of Low Temperature Response in Yeast Cells Using a cDNA Microarray

  • Changes in expression of genes in yeast after cold shock was analyzed using cDNA of 5,803 genes in a yeast genome
  • Ratio of fluorescent intensities was 2 fold for a mainly all the cDNA spots on the data
  • Roughly 25% of yeast genes' expression levels were affected by cold shock
  • Increase in number of genes up-regulated
  • Increase in number of genes down-regulated
  • Significantly up or down-regulated genes were classified according to the MIPS functional database
  • Up-regulated and down-regulated genes increased in almost all categories when exposed to low temperatures
    • Suggests that changes in gene expression are due to the introduction of low temperatures in order to adapt to their environment
    • Other cells have demonstrated this ^^ when exposed to other environmental stresses

Clustering Analysis of Global Expression Data

  • Genes close together were placed into clusters
  • Clusters showed:
    • Similar functions
    • Cooperative regulation
  • Clusters:
    • 1A: Unclassified proteins
    • 1B: Amino acid biosynthesis and metabolism
    • 1C: RNA polym. I & RNA processing
    • 1D: Ribosomal proteins
    • 1E: Not defined
  • Up regulated genes were divided into three clusters depending on their expression profiles
    • IC: up-regulated after within 30 mins after cold shock
    • ID: high up-regulation at 2hr and at 4-8 hr
    • IE: high up-regulation at 2hr and at 4-8 hr

Genes related to transcription

  • Further classification of genes:
    • 2A: RNA polymerase I & RNA processing - up regulated, then down regulated in later phase
    • 2B: rRNA processing - up regulated, then down regulated in later phase
    • 2C mRNA transcription - up-regulated in mid phase
    • 2D: mRNA transcription - continuously down regulated
  • These findings suggest that...
    • The mechanisms for transcription are up regulated when exposed to low temperatures in the early phase
      • Up-regulated mRNAs = essential for basic transcriptional/translational functions, like encoding regulatory proteins for amino acid production
    • The genes for transcriptional regulation and mRNA synthesis made diverse responses in the late phase
      • Down-regulated mRNAs = not essential for survival in cold shock, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes

Ribosomal Protein Genes

  • Cluster Classification:
    • 3A & 3B: cytosolic ribosome - up regulated first, then down regulated in later phase
    • 3C: translational control factors - continuously up regulated
    • 3D: tRNA synthetases - continuously down regulated
  • These findings suggest that...
    • Low temperature impairs translational ability
    • Yeast genes up-regulate to compensate

Cell Rescue, Defense, Death, and Aging

  • Cluster Classification:
    • 4A: not labeled
    • 4B & 4C: stress response - high up-regulation in mid-late phase
    • 4D: stress response and chaperone - high down-regulation in mid-late phase
  • These findings suggest that...
    • Heat shock protein genes down-regulated (EXCEPT for HSP12 and HSP26)
    • Protein folding genes up-regulated

Metabolism and Energy Production

  • Cluster Classification:
    • 5A: nucleotide metabolism - up-regulated early-mid phase, then down-regulated
    • 5B & 5E: not specified
    • 5C & 5D: C-compound and carbohydrate metabolism - continuously up-regulated
    • 5F & 5H: amino acid metabolism - continuously down-regulated
    • 5G: C-compound and carbohydrate utilization - continuously down-regulated
  • These findings suggest that...
    • Glycogen and trehalose production genes showed a lot of cooperative up-regulation
    • Trehalose may help protect cellular membrane, which may help to keep yeast cells intact at low temperatures

Signal Transduction Components

  • Cluster Classification:
    • 6A: signal transduction - down-regulated
    • 6B: signal transduction - up regulated in early phase, then down-regulation
    • 6C: signal transduction - down-regulated in early phase, then up-regulation in late
  • These findings suggest that...
    • Genes related to cAMP-PKA pathway and Msn2p/4p were up-regulated
    • Increase in PKA signaling has been known to be response to stresses
    • Increase in...
      • Signaling
      • Metabolism control
      • Stress resistance

Conclusion

  • Gene expression changed to maintain transcription and translation and to adapt a tolerance to the colder temperature
  • Transcriptional genes up-regulated first to help transcription and translation
  • Ribosomal proteins up-regulated in the middle phase to further assist maintenance of translation
  • Stress response induced genes up-regulated in the late phase
  • Maintaining translation is priority to yeast in cold shock
  • Cells need to make proteins to help maintain integrity and basic functions of cells
  • Yeast cells can adapt to environment once ^^^ obtained to gain tolerance to low temperature
  • Other organisms show similar responses in gene expression when exposed to environmental stresses

Deliverable

Journal Club Week 12 Presentation

Acknowledgements

I met with Corrine outside of class to discuss the method of presenting for the journal club. We worked on our presentation together and equally prepared for the assignment. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.
Dbashour (talk) 11:57, 21 November 2017 (PST)

References

Dina Bashoura

Biological Databases Homepage

List of Assignments

List of Individual Journal Entries

List of Shared Journal Entries

List of Final Assignments

List of Team Journal Assignments

Retrieved from "https://xmlpipedb.lmucs.io/biodb/fall2017/index.php?title=Dbashour_Week_12&oldid=5093"