This webpage was produced as an assignment for an undergraduate course at Davidson College.
PROTEOMICS VIEW OF BUD8
Here I am presenting information I have gathered, or tried to gather, from the proteomics databases available on the same genes used for assignments two and three.
Protein sequence:
miqsdednld ssettastsy sgtssvssrl qlrtslffen lngahgnpda etematvaye
ttsrgqgfav yinnerfsqi mgastsssss snsssitqfh dtqdnnipsn ttvrptslrr
dnedtvplrn vtpsqnaavr peravnspss qrlscaltis tsvlmgedve gspieqehsr
vvsslyssla nrgndeskng tpprptsiep nettehsffs yhyddtlepd veeavrltkn
ktsnvnfiss tgskgegete devidqyepv neskfiphkl kipekagsik sstsddshsp
gapgtsarki kipqspslig nilipshnsd ssnesspkdh ighnneekfs skstrkpsts
leeegppigl psipvlrsvs gpskwtktpl rlesgnstks dpfsryeghk tpspltkmnk
kknktlpehg qplvlapiks qssesdtgqn siiekparsi rrkqqektdn rkedrhdaen
idlearmpiq hidtasihsf dsgqngfrdv ysieniivil lccsivpplf fiigcssrrk
lvsdyrlmrl lmnkehraal lqgfiwdvdl rwfrmfclil gaaetvivma giaigfgvgi
tre
DATABASE OF INTERACTING PROTEINS (DIP)
Bud8 is the red dot in the middle of the figure. The four lines connecting to Bud8 also connect to the proteins that have been found to interact with Bud8.
FOUR GENES THAT INTERACT WITH BUD8:
N116 is a nuclear pore complex protein. (Not found on SGD Database)
YBN7 is a probable membrane protein. (Not found on SGD Database)
STE20 is probable serine/threonine specific protein kinase. This protein kinase is involved in pheromone response and pseudohyphal growth pathways and is located on chromosome VIII. ( http://genome-www.stanford.edu/Saccharomyces/)
YKI2 is a hypothetical protein.
http://dip.doe-mbi.ucla.edu/
All of the following data can be found at FUNCTION JUNCTION:
Go to:
http://genome-www.stanford.edu/cgi-bin/SGD/Sacch3D/getblast?name=BUD8/YLR353W&db=yeast&query=yeast for BLASTPed data against other yeast protein sequences.
The SGD SAGE Query produced the following information. For more information click on the links.
TAG CATGTACCCGTAAG
COORD MAP LINK: 836278
YGAC Triples Database - http://ygac.med.yale.edu/triples/triples.htm, had some information from mTn experiments with BUD8.
DATABASES UNABLE TO PROVIDE ANY INFORMATION:
GenTHREADER http://insulin.brunel.ac.uk/psipred/
PDB files http://www.rcsb.org/pdb/index.html
Function Junction, has several useful links and a compilation of information found from proteomics databases, some of which is highlighted below.
Yeast PathCalling - http://portal.curagen.com/extpc/com.curagen.portal.servlet.Yeast showed that YLR352W interacts with SKP1. SKP1 is found on chromosome IV and is a ubiquitin-protein ligase. It's biological process is G1/S and G2/M transition of mitotic cell cycle and also ubiquitin-dependent protein degradation. SKP1 is involved in kitetochore function and ubiquitin-mediated proteolysis. SGD Database
The SGD SAGE Query produced the following information. For more information click on the links.
TAG CATGATTTACGTGA
COORD MAP LINK 833113
DATABASE OF INTERACTING PROTEINS (DIP):
At the DIP site YLR352W (in the center) was found to interact with two annotated proteins and one non-annotated protein. SKP1, as before mentioned, and VATX, an H+ transporting ATPase are both annotated. The non-annotated protein is YDR372c.
DATABASES UNABLE TO PROVIDE ANY INFORMATION:
YGAC Triples Database - http://ygac.med.yale.edu/triples/triples.htm
GenTHREADER http://insulin.brunel.ac.uk/psipred/
PDB files http://www.rcsb.org/pdb/index.html
EXPERIMENT 1:
The first experiment I would perform to define the interactions and functions of YLR352W would be a Yeast Two-Hybrid method (Y2H). Briefly this method uses bait protein, in this case YLR352W, attached to half of a transcription factor called the DNA Binding Domain. In another cell, the other half of the transcription factor (the Activation Domain) is fused to various prey cells which may or may not interact with the bait protein. Transcription of the reporter gene can only occur if the DBD and AD interact because of the interactions of the proteins. We already know that SKP1, VATX, and YDR372c interact with our gene of interest. Good candidates for possible interaction could maybe be TPS2, ATH1, NTH1, or BCY1 which showed similar expressions as YPR352W in microarray experiments. The whole yeast proteome could be used in this analysis to see which proteins interact with YLR352W. Cells without the reporter gene, such as HIS3, cannot survive on medium without histidine, so if the proteins interacted then there will be a visible culture when the cells are grown on plates lacking histidine. (Campbell 462)
EXPERIMENT 2:
In assignment two I predicted that the YLR352W protein could possibly have something to do with stress response because it clustered with several different genes that were known to be involved in stress response. To test this prediction, one could do a series of gene knockout experiments with the genes that are known to function in stress response. If exposed certain stressful conditions such as heat shock, chemical exposure, amino acid starvation, osmotic shock etc., the cell could then be assessed for its response. This would be a good way to tell if the knockout genes are essential or not, and which ones need to be activated under the same conditions.
NCBI Database http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=AAB67749.1&form=6&db=p&Dopt=g, accessed November 7, 2001.
DATABASE OF INTERACTING PROTEINS (DIP) http://dip.doe-mbi.ucla.edu/, accessed November 7, 2001.
Function Junction http://genome-www.stanford.edu/cgi-bin/SGD/functionJunction?locus=BUD8, accessed November 7, 2001.
YGAC Triples Database - http://ygac.med.yale.edu/triples/triples.htm, accessed November 7, 2001.
Campbell, A. Malcolm. "Chapter Six: Proteomics." Genomics, Proteomics, and Bioinformatics. 2001.
EMAIL ME AT mrwilson@davidson.edu