Examining Microarray Experiments to Further Determine the Roles of my Favorite Yeast Genes: FUS1 and YCLO48W

 

"This web page was produced as an assignment for an undergraduate course at Davidson College."

 

 

Overview

 

In the previous assignment, I began to study two yeast genes, one of which was annotated, the other of which was not. This web page is a continuation of that exploration. The following is an assessment of the information I have gathered regarding each gene using microarray data from the Expression Connection web site at Stanford University.  The microarray data was chosen because it further establishes the role of the annotated gene in the cell and provides additional information regarding the non-annotated gene. 

 

 

Below is the color scale of fold repression/induction, which will be the standard scale used throughout this web page.

 

Expression at different alpha-factor concentrations (Rosetta Inpharmatics).

Orf

 

Gene

 

 

Process

 

Function

 

Component

YCL027W

 

FUS1

 

 

conjugation (sensu Saccharomyces)

 

molecular_function unknown

 

plasma membrane

YNL279W

 

PRM1

 

 

cell-cell fusion

 

molecular_function unknown

 

integral membrane protein*

YCR089W

 

FIG2

 

 

mating (sensu Saccharomyces)

 

not yet annotated

 

not yet annotated

YBL016W

 

FUS3

 

 

protein amino acid phosphorylation*

 

MAP kinase

 

nucleus*

 

* : indicates that more than one annotation exists for the gene.

See the Summary of the Gene Ontology annotations for this gene group

 

From the above microarray data, one can see that the gene PRM1, found on chromosome XIV, responds to differing concentrations of alpha-factor in a similar way as FUS1.  The two genes are similar in several ways. Both the FUS1 and the PRM1 are membrane proteins.  The FUS1 has a plasma membrane component and the PRM1 is an integral membrane protein. Both are found in the shmoo tip. PRM1 is involved in cell-cell fusion which is a component of conjugation. The null PRM1 mutant is viable much like the FUS1 mutant, and in both cases the yeast experiences a mating defect with a mutation.

 

The FUS1 also clusters with an ORF that is not annotated. The non-annotated gene FIG2 is found on the same chromosome as FUS1, chromosome III, and is also involved in mating.  The transcription of this gene is induced by the mating pheromones, a and alpha factor. The gene is required for efficient mating just as FUS1 is required for efficient mating as show because a mutation in FUS1 leads to ineffective cell fusion during mating.

 

FUS1 clusters with FUS3 when the microarrays are based on transcription at varying alpha-factor concentrations.  The similarity between FUS1 and FUS3 will be described below in the clustering of microarrays based on transcription in response to alpha-factor over time.

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl#alpha_conc)

 

 

Expression in response to alpha-factor (Rosetta Inpharmatics).

Orf

 

Gene

 

 

Process

 

Function

 

Component

YCL027W

 

FUS1

 

 

conjugation (sensu Saccharomyces)

 

molecular_function unknown

 

plasma membrane

YBL016W

 

FUS3

 

 

protein amino acid phosphorylation*

 

MAP kinase

 

nucleus*

YLR452C

 

SST2

 

 

signal transduction*

 

GTPase activator

 

plasma membrane

YNL279W

 

PRM1

 

 

cell-cell fusion

 

molecular_function unknown

 

integral membrane protein*


* : indicates that more than one annotation exists for the gene.

See the Summary of the Gene Ontology annotations for this gene group

 

In this microarray data, FUS1 groups with three other genes that all have common microarrays.  FUS1 clusters closely with FUS3, which is found on chromosome II. The protein is found in the nucleus which seems unrelated to the location of the FUS1 protein. Yet the function of this gene proves to be quite relevant to FUS1.  FUS3 is involved in signal transduction of the mating signal. The protein is required for cells to arrest in the G1 stage of the cell cycle in response to pheromone release and fusion of cells during conjugation.  

 

FUS1 also pairs with the gene SST2 which is found on chromosome XII. This gene product is found in the plasma membrane and is involved in the cell's ability to adapt to the mating signal. In other words, SST2 allows the cell to desensitize to the alpha-factor pheromone. Finally, FUS1 appears to have similar transcription in response to alpha-factor as PRM1 which has already been described.

 

Because these genes have such similar expression profiles, perhaps they are all part of one pathway for cell fusion during conjugation.  This pathway includes the detection of pheromones, the arrest of the cell cycle in G1, and the fusion of the two mating cells.

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl#alpha_conc)

Expression during the cell cycle  (Stanford University).

Name

Score

Peak

FUS1

1.71

M/G1

Reference Genes

 

CLN2

10.9

G1

HTA1

10.68

S

CLB4

3.08

G2

SWI5

6.726

M

ASH1

11.8

M/G1

 

 

 

 

Plot of FUS1 (YCL027W)

 

FUS1 does not appear to be very similar to any particular reference gene according to this microarray.  This data illustrates the response of FUS1 with respect to different experimental conditions and then this response can be compared to reference genes chosen because of their peak behavior at different points in the cell cycle. 

 

FUS1 has peak behavior during the M and G1 phase of the cell cycle.

 

With the introduction of the alpha mating pheromone, there is little response of FUS1.  Alpha pheromone does cause some gene transcription initially and then there is for the most part tapering off of any activity.  This makes sense if FUS1 is more closely related to the process of acclimation to the mating pheromones or if FUS1 is not part of the original signal pathway.  The point in which this gene is repressed is most similar to CLN2 which peaks at G1.  

 

It appears from the data that FUS1 is most active in mutant strain cdc15.  Most likely this strain arrests in the S phase.  The FUS1 gene has a similar expression profile yet there is strong activation in the end which is unlike all of the reference genes.   

 

The graphs further illustrate the response of FUS1 with peaks above the X axis representing a strong activation and peaks below the axis representing suppression.

 

 

Name

Score

Peak

FUS1

1.71

M/G1

SST2

2.406

M/G1

 

When submitting the FUS1 profile to obtain additional genes that cluster with this, the most notable gene is the SST2 which has a very similar expression profile.  This gene is described in the section above. (http://genome-www.stanford.edu/cgi-bin/cellcycle/drawCCquery.pl)

 

 (http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl#alpha_conc)

 

Expression in response to histone depletion  (The Whitehead Institute).

Orf

 

Gene

 

 

Process

 

Function

 

Component

YCL027W

 

FUS1

 

 

conjugation (sensu Saccharomyces)

 

molecular_function unknown

 

plasma membrane

YLR451W

 

LEU3

 

 

leucine biosynthesis

 

transcription factor

 

not yet annotated

YDR387C

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

 

See the Summary of the Gene Ontology annotations for this gene group

 

This microarray data shows that FUS1 clusters with two non-annotated genes.  This is not unlike many of the other microarray experiments in which the nearest genes are either not annotated or have no known information about them. There is no obvious relationship between FUS1 and LEU3.  LEU3 is involved in the expression of genes involved in branch chain amino acid synthesis and ammonia assimilation.

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl#alpha_conc)

 

Non-Annotated Gene YCL048W

 

Expression in response to alpha-factor  (Rosetta Inpharmatics).

Orf

 

Gene

 

 

Process

 

Function

 

Component

YCL048W

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YDL214C

 

PRR2

 

 

MAPKKK cascade

 

receptor signaling protein serine/threonine kinase

 

cellular_component unknown

YOL006C

 

TOP1

 

 

transcription regulation, from Pol II promoter*

 

DNA topoisomerase I

 

nucleus

* : indicates that more than one annotation exists for the gene.

See the Summary of the Gene Ontology annotations for this gene group

 

The PRR2 gene is a receptor signaling protein.  The gene product of this protein is a protein kinase.  The gene TOP1 is related to DNA elongation and chromatin assembly which occurs during meiosis. This leads to the idea that the non-annotated gene I have chosen, which is described as a hypothetical ORF, is involved in mating or the signaling of conjugation. 

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl?query=SGS1&noSearch=1)

Expression during the cell cycle  (Stanford University).

Name

Score

Peak

YCL048W

0.298

 

Reference Genes

 

CLN2

10.9

G1

HTA1

10.68

S

CLB4

3.08

G2

SWI5

6.726

M

ASH1

11.8

M/G1

 

 

 

 

Plot of YCL048W

 

 

According to the data generated, there were no genes that aggregated with YCL048W when submitting the profile on the Stanford website. (http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl?query=SGS1&noSearch=1)

 

There is no peak information given for YCL048W.  The expression profiles are all fairly nonexpressive but for the expression profile in the cdc15 strain.  This microarray data seems to be similar to the profile for the reference gene CLB4 which peaks during G2. 

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl?query=SGS1&noSearch=1)

 

Expression in response to histone depletion  (The Whitehead Institute).

Orf

 

Gene

 

 

Process

 

Function

 

Component

YCL048W

 

 

 

 

biological_process unknown

 

molecular_function unknown

 

not yet annotated

YGL018C

 

JAC1

 

 

aerobic respiration

 

cochaperone

 

mitochondrion

YMR182C

 

RGM1

 

 

Transcription

 

not yet annotated

 

not yet annotated

YOR181W

 

LAS17

 

 

cytokinesis*

 

cytoskeletal protein binding protein

 

actin cortical patch (sensu Saccharomyces)


* : indicates that more than one annotation exists for the gene.

See the Summary of the Gene Ontology annotations for this gene group

 

The JAC1 protein is involved in the assembly of mitochondrial iron-sulfur proteins for the cell process of aerobic respiration.  The RGM1 gene is involved with the process of repressing transcription though this gene is not yet annotated.  The LAS17 gene seems to be involved in polar budding and the gene product is an actin assembly factor.      

 

(http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl?query=SGS1&noSearch=1)

 

Conclusions

 

It can be concluded that much of the microarray data for the non-annotated gene YCL048W is inconclusive.  The other genes that this gene clusters with are not consistent as with the annotated gene FUS1 that clustered repeatedly with certain genes.  This helps to explain why the YCL048W gene is in fact not yet annotated.  The microarray data does not reveal much specific information with regard to this gene’s function or process.

 

This web page is a good example of the information that can be obtained using microarray data.  There is some amount of information accessible for every ORF even if it is not yet annotated.

 

 

 

 

References

 

Expression Connection <http://genome-www4.stanford.edu/cgi-bin/SGD/expression/expressionConnection.pl> Accessed 17 October 2001.

 

Lodish, H., A. Berk, S.L. Zipursky, P. Matsudaira, D. Baltimore, J. Darnell. Molecular Cell Biology. 2000. New York: W.H. Freeman and Co.

 

 

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