20.109(F08):Module 2: Difference between revisions
No edit summary |
|||
| Line 9: | Line 9: | ||
'''TA:''' [http://openwetware.org/wiki/User:Brian_Belmont Brian Belmont] | '''TA:''' [http://openwetware.org/wiki/User:Brian_Belmont Brian Belmont] | ||
Faced with the considerable challenge of packing more DNA into a cell, nature added proteins that reversibly compact the helix. The DNA can wind around these histone proteins, forming nucleosomes, that can then wind around each other to form chromatin. Other protein complexes modify and remodel the chromatin, making the DNA accessible for reading and copying. “SAGA” is a chromatin remodeling complexes in the model experimental yeast, S. cerevisiae, but it turns out not every protein in SAGA is needed for the yeast to survive. In this experiment we will modify one of the SAGA-subunits in the yeast genome and then ask how the resulting yeast, though alive, is affected. We will look for phenotypes that might indicate crippled functions and we will compare gene expression in the parent strain to each tagged strain using a microarray. Our individual experiments may identify | Faced with the considerable challenge of packing more DNA into a cell, nature added proteins that reversibly compact the helix. The DNA can wind around these histone proteins, forming nucleosomes, that can then wind around each other to form chromatin. Other protein complexes modify and remodel the chromatin, making the DNA accessible for reading and copying. “SAGA” is a chromatin remodeling complexes in the model experimental yeast, <i>S. cerevisiae,</i> but it turns out not every protein in SAGA is needed for the yeast to survive. In this experiment we will modify one of the SAGA-subunits in the yeast genome and then ask how the resulting yeast, though alive, is affected. We will look for phenotypes that might indicate crippled functions and we will compare gene expression in the parent strain to each tagged strain using a microarray. Our individual experiments may identify genes controlled by particular SAGA subunits while our class data may reveal genes that are commonly regulated by this remodeling complex. Given the structural information for SAGA that is recently available, we can hope to map our findings onto the complex and better understand the delicate balance between chromatin remodeling and gene expression. | ||
[[Image:Macintosh HD-Users-nkuldell-Desktop-ExpressionEng coverart S07.jpg|thumb|500 px|center| SAGA image from F.Winston<br> Mode of action model from P. Schultz <br> Mol Cell. 2004 Jul 23;15(2):199[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=4&itool=pubmed_docsum]]<br> Microarray image from N. Kuldell<br>]] | [[Image:Macintosh HD-Users-nkuldell-Desktop-ExpressionEng coverart S07.jpg|thumb|500 px|center| SAGA image from F.Winston<br> Mode of action model from P. Schultz <br> Mol Cell. 2004 Jul 23;15(2):199[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=4&itool=pubmed_docsum]]<br> Microarray image from N. Kuldell<br>]] | ||
Revision as of 17:48, 14 August 2008
pic.jpg)
Module 2
Instructors: Natalie Kuldell, Agi Stachowiak
TA: Brian Belmont
Faced with the considerable challenge of packing more DNA into a cell, nature added proteins that reversibly compact the helix. The DNA can wind around these histone proteins, forming nucleosomes, that can then wind around each other to form chromatin. Other protein complexes modify and remodel the chromatin, making the DNA accessible for reading and copying. “SAGA” is a chromatin remodeling complexes in the model experimental yeast, S. cerevisiae, but it turns out not every protein in SAGA is needed for the yeast to survive. In this experiment we will modify one of the SAGA-subunits in the yeast genome and then ask how the resulting yeast, though alive, is affected. We will look for phenotypes that might indicate crippled functions and we will compare gene expression in the parent strain to each tagged strain using a microarray. Our individual experiments may identify genes controlled by particular SAGA subunits while our class data may reveal genes that are commonly regulated by this remodeling complex. Given the structural information for SAGA that is recently available, we can hope to map our findings onto the complex and better understand the delicate balance between chromatin remodeling and gene expression.
Mode of action model from P. Schultz
Mol Cell. 2004 Jul 23;15(2):199[[1]]
Microarray image from N. Kuldell
Day 1: Protein engineering with PCR
Day 2: Yeast transformation
Day 3: Colony PCR, Journal article discussion
Day 4: SDS-PAGE, screen for phenotypes
Day 5: Probe Western, isolate RNA
Day 6: Journal Club I
Day 7: cDNA synthesis and microarray
Day 8: Microarray data analysis
direct link to writeup
direct link to oral presentation
