This web page was produced as an assignment for an undergraduate course at Davidson College.
As noted on the website created for my second assignment, CMD1 encodes for the protein calmodulin in Saccharomyces cerevisiae. Calmodulin is a ubiquitously expressed Ca2+ binding protein that interacts with the components of numerous cellular processes. Some of the processes that CMD1 is involved with are cytoskeleton organization, cell growth, cell division, and budding.In order to learn more about the role of CMD1 in yeast, I have consulted the Saccharomyces Genome Database's Expression Connection Search. Expression Connection is a powerful site that summarizes the expression profiles of genes as observed in a number of DNA microarrary experiments. Another interesting feature is that Expression Connection groups the expression profile of your gene with a number of other genes that have similar expression profiles.
Following the discussion of CMD1's expression, there is a similar discussion of an unannotated ORF, named YBR108W.
This is the scale used throughout the following figures for repression and induction:
(SGD, 2002; <Expression Connection>)
Expression in response to alpha-factor for CMD1/YBR109C (Rosetta Impharmatics)
Figure 1. Screen capture from alpha-factor exposure experiment (SGD, 2002; Expression Connection)
In this experiment, CMD1 becomes increasingly induced as it is exposed to a constant amount of alpha-factor over a duration of time. It is most closely grouped with the gene ARP2, a gene involved in actin filament organization. Gene ontology lists the function of ARP2 as a structural constituent of the cytoskeleton. This pairing is interesting because one of the many processes that CMD1 is involved in is organization of the cytoskeleton. CMD1 has also been grouped with PTP2, a gene involved in signal transduction of mating signal. Mating signal is a peptide pheromone that binds to a particular G-protein receptor which begins a cascade that results in induction of mating process-related genes (SGD, 2002; <Gene Ontology: Signal transduction of mating signal>). CMD1 participates in several transduction pathways including stress and pheromone response, so it not a surprise that these two have similar expression profiles (SGD, 2002; <Function Junction>; Cyert, 2001). Another noteworthy gene is ENT3, a cytoskeletal adaptor that acts in endocytosis. CMD1 has been shown to have some role in endocytosis, as some types of CMD1 mutants have exhibited defects in that process (Cyert, 2001). Figure 2provides a graphical depiction of CMD1 induction in this experiment, as the dark red can be hard to see. It is understandable that alpha-factor exposure could bring about the induction of CMD1, since alpha-factor is a mating pheromone and CMD1 is known to be involved with pheromone signal transduction (Cyert, 2001).
Figure 2: Increased expression of CMD1 due to alpha-factor exposure over 120 minutes (SGD, 2002; Expression Connection).
Expression during the diauxic shift for CMD1/YBR109C (Stanford University)
Figure 3. Screen capture diauxic shift experiment (SGD, 2002; Expression Connection)
The diauxic shift is the transition from anaerobic to aerobic respiration. In this experiment, the increase in expression of CMD1 remains nearly imperceptible until the 18.5 hour mark, where it is expressed very slightly. It has been grouped with two genes involved in cell wall organization and biogenesis: ECM25 and ROT1. Another aspect of this profile that interests me is the presence of 3 genes involved in ubiquitin-dependent protein catabolism: RPT2, PRE1, and PRN12. In addition to appearing on the diauxic shift expression profile, proteins for ubiquitin-dependent protein catabolism are grouped with CMD1 in the expression profiles for exposure to alpha-factor, exposure to DNA-damaging reagents, and expression during sporulation. CMD1 is grouped again with RPT2 in the DNA-damaging reagents experiment. There is no mention of a relationship between CMD1 and ubiquitin-dependent catabolism in the Cyert paper or on Function Junction. However, the fact that CMD1 is repetitively grouped with ubiquitin-related genes suggests that a relationship might exist. Since processes involving ubiquitin and calmodulin are both very common in yeast cells, it is plausible that their paths might cross at some point. One gene that Function Junction directly links to CMD1 is MYO2, which is involved in establishment of cell polarity. APB1, TPM2, located towards the bottom of the list, both encode for actin-binding proteins that participate in establishment of cell polarity. According to SGD, the diauxic shift experiment should identify genes that are involved in the anaerobic-aerobic transition (SGD Help, 2002; <Expression Help>. CMD1's lack of profound induction is consistent with the fact that involvement in diauxic shifts are not mentioned in the Cyert paper. However, the fact that induction occurs slightly over time suggests that it might be involved to some extent.
Expression in response to DNA-damaging agents for CMD1/YBR109C (Stanford University)
Figure 4. Screen capture from DNA-damaging agent experiment (SGD, 2002; <Expression Connection>)
Here, the expression profile for CMD1 in response to various DNA-damaging agents shows that CMD1 is induced very subtly throughout, especially in response to methyl methane sulfonate. The feature of highest induction is labeled 'mec1 mutant - log phase (irradition time=0 cells).' It has been grouped with four genes whose processes have been classified as ubiquitin-dependent protein catabolism. One of these, RPT2, was seen in the diauxic shift experiment above. Repression appears to be occurring at 'mec1 mutant + mock irradiation (5 min).'
Expression in response to environmental changes for CMD1/YBR109C (Stanford University)
Figure 5. Screen capture from environmental change experiment (SGD, 2002; <Expression Connection>)
This profile shows expression of CMD1 with respect to a number of different environmental stresses, including heat shock, temperature shifts, amino acid starvation, and exposure to chemicals like hydrogen peroxide, menadione, and DTT. Since CMD1 is known to play a part in the yeast cell's response to environmental stress, one would expect that CMD1 would exhibit induction during the various treatments. This indeed seems to be the case, although the induction was identified by bright red in only one instance. The color bar shows that induction of CMD1 occurred because of heat shock, menadione exposure, DTT exposure, diamide exposure, and after six hours of amino acid starvation. The greatest induction occurs throughout exposure to hydrogen peroxide, with the most profound induction occurring at the 40 minute mark. Repression also occurred, particularly after 100 minutes of hydrogen peroxide exposure, after one hour of amino acid starvation, during the phase labeled 'YPD Stationary Phase,' and when the YP medium was altered with ethanol and mannose as the carbon source.
Expression during the cell cycle for CMD1/YBR109C
(Stanford University, Cold Spring Harbor)
Figure 6. Screen capture from cell cycle experiment (SGD, 2002; <Expression Connection>)
This experiment looks at the expression of CMD1 throughout the cell cycle. At several points, it is induced or repressed, but ultimately, the score of 0.09 indicates that CMD1 is most likely not cell cycle regulated (Yeast Cell Cycle Analysis Project, 2000; <YCCAP>). Here is a link to a list of genes that were known to be cell cycle regulated prior to this experiment: Known Regulated Genes (Microsoft Word document). Use the find feature to search for CMD1, and you will see that it is not on this list.
Expression in response to histone depletion for CMD1/YBR109C (The Whitehead Institute)
Figure 7. Screen capture from histone depletion experiment (SGD, 2002; <Expression Connection>)
Histone depletion experiments identify genes whose transcription patterns are disrupted by histone depletion (SGD Help, 2002; <Expression Help>). The profile for CMD1 shows that repression has begun to occur at one hour, reaches peak repression at two hours, decreases at 4 hours, and then is on the increase again at 6 hours.
Expression during sporulation for CMD1/YBR109C (UCSF, Stanford University)
Figure 8. Screen capture from sporulation experiment (SGD, 2002; <Expression Connection>)
This experiment shows the expression of CMD1 with respect to the occurrence of sporulation. CMD1 has been grouped with a gene of unknown ontological process and function called MLC2. Their similar expression patterns suggest that they might be related in some way. At the SGD site for MLC2, I learned that the putative protein product of MLC2, Mlc2p, is most likely the light chain for Myo1p, type II myosin (SGD, 2002). MYO1 is involved in a many of the same processes as CMD1: cell growth, cell division, cell polarity, and organization of the cytoskeleton (SGD, 2002; <Function Junction: MYO1>).
Expression of YBR108W in S. cerevisiae
For our second assignment, we were asked to take an unannotated ORF, located near our annotated gene, and use public databases to discover as much as possible about the role of the unannotated ORF in S. cerevisiae. Although I found some interesting information about the ORF I chose, YBR108W, the data did not provide any conclusive evidence for its function, process, or cellular component, other than the fact that it might encode a globular protein.
Using Expression Connection, just as above, it is my hope that this might shed further light upon YBR108W, and its role in the life of S. cerevisiae.
I have reproduced the scale again for your convenience:
(SGD, 2002; <Expression Connection>)
Expression in response to alpha-factor for YBR108W (Rosetta Impharmatics)
Figure 9. Screen capture from alpha-factor experiment (SGD, 2002; <Expression Connection>)
In response to alpha-factor exposure, YBR108W becomes increasingly induced over time. Predominantly, YBR108W is grouped with unannotated genes. It has been grouped with another gene of unknown process, function, and cellular component. However, this particular gene, unlike YBR108W, has a specific gene name: PAU6. SGD describes PAU6 as a member of the seripauperin protein/gene family (2002; <SGD: PAU6>). I searched SGD for PAU1-5, and they all have the same nebulous description. A search of the SGD for 'seripauperin' turned up no results, so I tried PUBMED. This search also yielded no hits. Finally I tried Google, where a number of hits came up. Although most of the hits gave any more information than SGD, some of the hits referred to the PAU genes as possible cell wall proteins (Fowlkes, 2002; <http://www.cs.berkeley.edu/~fowlkes/bio/clusters/final1/depth4/gcluster2.html>). Interestingly enough, 3 lines down from PAU6 is CAP1, whose process is listed as cell wall organization and biogenesis. Throughout the various experiments below, YBR108W is grouped with other cell wall organization genes, but as closely as in this experiment.
Expression during the diauxic shift for YBR108W (Stanford University)
Figure 10: Screen capture from diauxic shift experiment (SGD, 2002; <Expression Connection>)
During the shift from anaerobic to aerobic respiration, it appears that YBR108W is increasingly repressed. Once again, it is mainly grouped with unknown genes. In this list of similar genes, there are 2 genes listed whose processes are cell wall organization and biogenesis.
Expression in response to DNA-damaging agents for YBR108W (Stanford University)
Figure 11. Screen capture from DNA-damaging agents experiment (SGD, 2002; <Expression Connection>)
Throughout the exposure to the various DNA-damaging agents, expression of YBR108W remains unchanged, as designated by the black portions of the color bar. Induction can be seen during exposure to gamma irradiation, with peak induction at the 45 minute mark. There also appears to be induction after 90 minutes of mock irridiation of wildtype cells, although it is unclear to me what exactly constitutes mock irradiation. The MEC1 mutant exhibited no induction of YBR108W throughout the 120 minutes of gamma irradiation.
Expression in response to environmental changes for YBR108W (Stanford University)
Figure 12: Screen capture from environmental stress experiment (SGD, 2002; <Expression Connection>)
Exposure to various environmental stresses seems to elicit a number of responses in YBR108W expression. The different heat shock experiments resulted in slightly more induction than repression, but it still seems like it should be more consistent. After 30 minutes of exposure to hydrogen peroxide, YBR108W is induced, but ten minutes later it is repressed. I would like to know more about what could cause such a turnaround. One thing about this profile that I found interesting was the extent of repression and induction that the ORF exhibits. The brightness of the color bar is much more pronounced than that of my annotated gene, CMD1. This is contrary to my expectations. CMD1 is known to be a component of signal transduction in response to environmental stress. Although it appears consistently induced in this same experiment, I would have expected it to exhibit larger amounts of induction. The red signals for YBR108W are much brighter, so perhaps it plays some part in response to environmental stress.
Expression during the cell cycle for YBR108W (Stanford University, Cold Spring Harbor)
Figure 13: Screen capture from cell cycle experiment (SGD, 2002; <Expression Connection>)
In looking at expression with regards to the various stages of the cell cycle, it appears that YBR108W's expression might be regulated by the cell cycle. Although a score of 2.777 is not very high, there is a better chance of YBR108W being regulated by the cell cycle than CMD1, which received a score very near to zero.
Expression in response to histone depletion for YBR108W (The Whitehead Institute)
Figure 14: Screen capture from histone depletion experiment (SGD, 2002; <Expression Connection>)
The level of YBR108W transcription appears to be disrupted by histone depletion, as the color bar shows that induction on a scale of nearly 2.8x is occurring. YBR108W has been grouped most closely with CYR1, a gene which is apparently involved with meiosis. YBR108W has also been grouped with two more cell wall organization genes: MYO3 and CNA1.
In conclusion, it seems that CMD1 was consistently grouped with genes to which it could plausibly be related. Although it was never linked to any of the genes that Function Junction and Pathcalling list as direct points of interaction (Fig. 15), in several instances it was grouped with genes related either closely or distantly to them. Finally, I also learned that GenomeNet places CMD1 in the phosphatidylinositol signaling system of yeast. Although the Cyert paper discusses some of CMD1's interactions with signalling pathways, this particular name never came up. Here is a link to a diagram of the pathway on the GenomeNet website: Phosphatidylinositol signaling system (GenomeNet, 2002; <GenomeNet>).
Figure 15: Screen capture of Pathcalling interactions of CMD1. According to this picture, it directly interacts with MYO2, NUF1, MYO5, and SLT2 (Genescape Portal Website, 2000; <Function Junction>).
At the conclusion of Assignment 2, I was unsure about the unannotated ORF YBR108W, but I predicted that if it did encode for something, it was probably a globular protein. From Expression Connection, I learned that YBR108W is expressed fairly often in response to various conditions imposed upon yeast cells. In fact, the intensity of the color bars were often more brilliant than the darker expressions that CMD1 tended to produce. YBR108W, over all, seemed to be most commonly grouped with unknown genes or genes involved in cellular organization and biogenesis. Therefore, it seems that YBR108W must play some role in the life of S. cerevisiae. However, I can only speculate that it might be either a cell wall protein or involved in the organization of the cell wall at this time.
"CMD1/YBR109C." Saccharomyces Genome Database. Last Update: 2002. <http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=cmd1>
Cyert, Martha S. "Genetic analysis of calmodulin and its targets in Saccharmoyces cerevisiae." Annual Review of Genetics. 35: 647-672 (2001).
Expressin Connection Search. Saccharomyces Genome Database. <http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl?query=CMD1&noSearch=1>
Expression Help. Saccharomyces Genome Database. <http://genome-www4.stanford.edu/Saccharomyces/help/expression.html>
Fowlkes, Charless. "Clustering of Rosetta Dataset." Genecut Homepage. 2002. <http://www.cs.berkeley.edu/~fowlkes/bio/clusters/final1/depth4/gcluster2.html>
Function Junction Search. Saccharomyces Genome Database. <http://genome-www4.stanford.edu/cgi-bin/SGD/functionJunction?locus=CMD1>
Genescape Portal Website. Curagen Corporation Website. 2002. <http://portal.curagen.com/>.
"Phosphatidylinositol signaling system." GenomeNet Home. <http://www.genome.ad.jp/>
"PAU6." Saccharomyces Genome Database. Last Update: 2002. <http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=pau6>
"YBR108W." Saccharomyces Genome Database. Last Update: 2002. <http://genome-www4.stanford.edu/cgi-bin/SGD/locus.pl?locus=ybr108w>
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