Difference between revisions of "Cwong34 Week 12"

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(Added acknowledgment and signature)
(Added to outline)
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=Flow Chart=
 
=Flow Chart=
 
=Outline=
 
=Outline=
 +
==Experiment design and procedures==
 +
*Purchased cDNA microarray of ''Saccharomyces cervisiae'' from DNA Chip Research Inc.
 +
*Used ''S. cervisiae''YPH500
 +
*Grew yeast cells aerobically in YPD medium at 30 degrees celsius and shaken at 100rpm.
 +
*YPD is made up of:
 +
**1% yeast extract
 +
**2% peptone
 +
**2% glucose
 +
*Yeast cells were grown to "mid-log phase" where they were still maturing, but not fully reproducing
 +
*50mL of culture was taken and centrifuged to collect the cells and be used as a time 0 reference for the rest of the experiment
 +
*The time 0 reference cells were stored at -80 degrees celsius
 +
*The rest of culture was used for the experimental samples and was cold shocked at 10 degrees celsius
 +
*Samples were collected at various times: 0.25, 0.5, 2, 4, and 8h
 +
*Acidic phenol method and RNeasy Mini Kit were used to prepare the RNA
 +
*The RNA was used to prepare fluorophore-labeled cDNA probes
 +
*These probes marked the cells for array hybridization
 +
*The microarrays were scanned with a laser microscope and were analyzed
 +
*Repeated process twice
 +
*Averaged the expression ratios of the separate experiments for final data
 +
==Results & discussion==
 +
*Analyzed the microarray of cDNAs of 5,803 genes in yeast genome
 +
*There was a diauxic shift in cells that experienced cold shock
 +
**Down-shift of some diauxic shift-inducible genes in late phase of cold shock
 +
*Low temperature affects expression of ~25% of the yeast genes
 +
*Number of up-regulated genes increased from 41 at 0.25h to 536 at 8h
 +
*Number of down-regulated genes also increased from 4 at 0.25h to 488 at 8h
 +
*Gene expression changes significantly in both ways (up & down-regulation) to adapt cells to colder environment, similar to reactions to other stresses, like heat, salinity, hydrogen peroxide, and osmotic stresses
 +
*Table 1 shows the number of genes that significantly changed expression (2-fold or more), up-regulating or down-regulating
 +
===Clustering analysis===
 +
*Clustering of genes (genes that were close together) were analyzed in the ways they responded
 +
*Genes separated into 5 different clusters (Fig. 1):
 +
**1A: Unclassified proteins
 +
**1B: Amino acid biosynthesis and metabolism
 +
**1C: RNA Polymerase I & RNA processing
 +
**1D: Ribosomal proteins
 +
**1E: Not labeled
 +
*Shows cooperative regulation
 +
**1C: up-regulated in early phase (0-0.5h), then down-regulated in late phase (4-8h)
 +
**1D & 1E: up-regulated in mid-late phase (2h & 4-8h)
 +
===Transcription related genes===
 +
*Looked at clusters of genes relating to RNA polymerase I & RNA processing
 +
**All up-regulated in early phase
 +
*Cooperative regulation of genes involved in transcription (Fig. 2)
 +
*2 clusters of these genes:
 +
**Down-regulating (2A, 2B, & 2D)
 +
***2A (RNA polymerase I & RNA processing) & 2B (rRNA processing) up-regulated in early phase, then down regulated in late phase
 +
***2D: mRNA transcription
 +
**Up-regulating (2C)
 +
***2C: mRNA transcription
 +
***High up-regulating in mid phase
 +
*Up-regulated genes that had to do with basic transcriptional functions, like genes encoding for regulatory proteins for amino acid production
 +
*Down-regulated genes were not essential for basic life, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes
 +
*Factors essential to transcription & processing of rRNAs were up-regulated
 +
*Genes for synthesis and transcription regulation of mRNAs had mix responses
 +
===Ribosomal protein related genes===
 +
*
 
=Terms=
 
=Terms=
 
#Nucleolin: a protein associated with nucleolar in growing eukaryotic cells (NCBI, 2017).
 
#Nucleolin: a protein associated with nucleolar in growing eukaryotic cells (NCBI, 2017).

Revision as of 02:49, 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.

Flow Chart

Outline

Experiment design and procedures

  • Purchased cDNA microarray of Saccharomyces cervisiae from DNA Chip Research Inc.
  • Used S. cervisiaeYPH500
  • Grew yeast cells aerobically in YPD medium at 30 degrees celsius and shaken at 100rpm.
  • YPD is made up of:
    • 1% yeast extract
    • 2% peptone
    • 2% glucose
  • Yeast cells were grown to "mid-log phase" where they were still maturing, but not fully reproducing
  • 50mL of culture was taken and centrifuged to collect the cells and be used as a time 0 reference for the rest of the experiment
  • The time 0 reference cells were stored at -80 degrees celsius
  • The rest of culture was used for the experimental samples and was cold shocked at 10 degrees celsius
  • Samples were collected at various times: 0.25, 0.5, 2, 4, and 8h
  • Acidic phenol method and RNeasy Mini Kit were used to prepare the RNA
  • The RNA was used to prepare fluorophore-labeled cDNA probes
  • These probes marked the cells for array hybridization
  • The microarrays were scanned with a laser microscope and were analyzed
  • Repeated process twice
  • Averaged the expression ratios of the separate experiments for final data

Results & discussion

  • Analyzed the microarray of cDNAs of 5,803 genes in yeast genome
  • There was a diauxic shift in cells that experienced cold shock
    • Down-shift of some diauxic shift-inducible genes in late phase of cold shock
  • Low temperature affects expression of ~25% of the yeast genes
  • Number of up-regulated genes increased from 41 at 0.25h to 536 at 8h
  • Number of down-regulated genes also increased from 4 at 0.25h to 488 at 8h
  • Gene expression changes significantly in both ways (up & down-regulation) to adapt cells to colder environment, similar to reactions to other stresses, like heat, salinity, hydrogen peroxide, and osmotic stresses
  • Table 1 shows the number of genes that significantly changed expression (2-fold or more), up-regulating or down-regulating

Clustering analysis

  • Clustering of genes (genes that were close together) were analyzed in the ways they responded
  • Genes separated into 5 different clusters (Fig. 1):
    • 1A: Unclassified proteins
    • 1B: Amino acid biosynthesis and metabolism
    • 1C: RNA Polymerase I & RNA processing
    • 1D: Ribosomal proteins
    • 1E: Not labeled
  • Shows cooperative regulation
    • 1C: up-regulated in early phase (0-0.5h), then down-regulated in late phase (4-8h)
    • 1D & 1E: up-regulated in mid-late phase (2h & 4-8h)

Transcription related genes

  • Looked at clusters of genes relating to RNA polymerase I & RNA processing
    • All up-regulated in early phase
  • Cooperative regulation of genes involved in transcription (Fig. 2)
  • 2 clusters of these genes:
    • Down-regulating (2A, 2B, & 2D)
      • 2A (RNA polymerase I & RNA processing) & 2B (rRNA processing) up-regulated in early phase, then down regulated in late phase
      • 2D: mRNA transcription
    • Up-regulating (2C)
      • 2C: mRNA transcription
      • High up-regulating in mid phase
  • Up-regulated genes that had to do with basic transcriptional functions, like genes encoding for regulatory proteins for amino acid production
  • Down-regulated genes were not essential for basic life, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes
  • Factors essential to transcription & processing of rRNAs were up-regulated
  • Genes for synthesis and transcription regulation of mRNAs had mix responses

Ribosomal protein related genes

Terms

  1. Nucleolin: a protein associated with nucleolar in growing eukaryotic cells (NCBI, 2017).
  2. Hypoxia: low levels of oxygen in body tissues (Hine & Martin, 2015).
  3. Ubiquitin: a protein that marks which proteins are going to be broken down (Hine & Martin, 2015).
  4. "Mid-log" or "lag" phase: bacterial cells are maturing, but they have not yet reached their maximum reproduction rate (Hine & Martin, 2015).
  5. Diauxic shift: the switch of a microorganism from using one type of sugar to using another (King, et al., 2014).
  6. Cytosolic: contents of the fluid in the cytoplasm of a cell (King, et al., 2014).
  7. Peptidyl proly cis/trans isomerases: Enzymes that changes conformation of prolyl bonds to cis or trans, chaning the tertiary structure of a protein (Lackie, 2013).
  8. cAMP-PKA pathway: the activation of PKA by cAMP (Lackie, 2013).
  9. GTPase: enzymes that hydrolyze GTP (Lackie, 2013).
  10. Phosphatase: an enzyme that helps remove a phosphate group from an organic compound (Hine & Martin, 2015).

Acknowledgments

  1. I met with Dina outside of class, and we worked on our presentation together.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. Cwong34 (talk) 17:28, 20 November 2017 (PST)

References

  1. Hine, R. & Martin, E. (Eds.). (2015). A Dictionary of Biology. In Oxford Reference. Retrieved from http://www.oxfordreference.com/view/10.1093/acref/9780198714378.001.0001/acref-9780198714378
  2. 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
  3. Lackie, J.L. (2013). The Dictionary of Cell and Molecular Biology. In Science Direct. Retrieved from http://www.sciencedirect.com/science/book/9780123849311
  4. LMU BioDB 2017. (2017). Week 12. Retrieved November 14, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12
  5. NCBI. (2017). Nucleolin. Retrieved November 20, 2017, from http://www.uniprot.org/uniprot/P19338
  6. 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.

cwong34

BIOL/CMSI 367-01: Biological Databases Fall 2017

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