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STUDIES ON PROTEIN KINASE

SIGNALLING PATHWAY INVOLVED IN


SCHIZOSACCHAROMYCES POMBE

M.SHANMUGA PRIYAA
M.Sc BIOTECHNOLOGY
GENOME INSTITUTE OF SINGAPORE
Comparative study, on the expression of protein
kinase genes pertaining to the signaling pathway of
the cell division cycle (cdc), of S.pombe in both the
wild and mutant strains is being investigated using
micro array technology.
• Schizosaccharomyces pombe-
fission yeast.

• Pombe is derived from the


Swahili word for beer.

• Isolated in 1893 by Lindner from


millet beer.

• The sixth model eukaryotic


organism whose genome has been
completely sequenced.

• S.pombe is,
an unicellular eukaryote,
rod-shaped
2 - 3 µm in diameter
7-14 µm in length
WILD MUTANT
CELL CYCLE :

INTERPHASE
G1-S-G2
M PHASE
PROPHASE,
METAPHASE,
ANAPHASE,
TELOPHASE.

Researches in cell cycle,helps to understand how cells grow and differentiate


as well as how the non-controlled growth results in cancerous cells.

Key players in the progression of the cell cycle are a group of kinases.
KINASES

• Binds to cyclins-regulate
cyclins kinase activity.
• Inactive kinases-cdk’s
• Add phosphate group
• Phosphorylation of proteins at different stages of the cell cycle is
necessary for the cycle to complete.
GENE EXPRESSION :
Complex pattern of expression
Determines the characteristics of a cell and its role in the organism.
Abnormal pattern- cancer.
MICROARRAY:
• To assay gene expression within a single cell.
• To compare gene expression in two different cell types or tissue samples.
• To examine the expression of hundreds or thousands of genes at once.
STEPS:
1.Preparation of DNA chips using known DNAs.
2.Generation of a hybridization solution containing a mixture of
fluorescently labeled cDNAs (mutant type).
3.Incubation of the hybridization mixture containing fluorescently labeled cDNAs with
DNA chip.
4.Detection of bound cDNAs using laser technology and storing data in a computer.
5.Analyzing and interpreting the data using computational methods.
Cell culture

Total RNA extraction

Reverse Transcriptional reaction

CDNA probe preparation

Coupling – CYS dyes


Preparation of Micro
array slides.

Spotting of array’s
Hybridization

Yellow =equal expression

Scanning
•The image analysis - 2 final intensity measurements for the red and green
are obtained for every single spot.

SCANNED SLIDE

Green represents - the expression of the control DNA (wild),


wild
Red represents - the expression of the Sample DNA (reference),
reference
yellow represents - the expression of both the control and sample
DNA.
Black represents - the expression of the neither the control nor
sample.
CLUSTERED IMAGE PRODUCED USING GENE PIX PRO4.1

Each row -- a gene Each column - a sample.


KO 42-pck1; KO 49-cdc2; KO 51-chk1; KO 55-fin1; KO 58-cmk1; KO 59-orb6;
KO 60-plo 1; KO 61-wis1; KO 62-spk 1; KO 65-cds1; KO 66-ssp1;
KO 67-mek1; KO 75-kin1; KO 76-cdc7; KO 77-wee 1; KO 81-win 1;
The pattern of expression of Protein kinase mutant
strain
Orb6 (KO-59 A &B)

Green color - a particular


gene in the mutant is suppressed
(down regulated).
Red color- a particular gene
in the mutant is induced (up
regulated).
GREY color - the missing data.
BLACK color - no expression

Each row -- a gene


The pattern of expression of Protein kinase mutant strain

Plo1 (KO-60 A &B)

Green color - a particular gene in


the mutant is suppressed.
Red color - a particular gene
in the mutant is induced.
GREY color - the missing data.
BLACK color - no expression
The pattern of expression of Protein kinase mutant strain
Wis1 (KO-61 A &B)

Green color - a particular gene in


the mutant is suppressed.
Red color - a particular gene
in the mutant is induced.
GREY color - the missing data.
BLACK color - no expression
The pattern of expression of Protein kinase mutant strain
Wee1 (KO-77 A &B)

Green color - a particular gene in


the mutant is suppressed.
Red color - a particular gene
in the mutant is induced.
GREY color - the missing data.
BLACK color - no expression
Tree view of Protein kinase mutants

The length of the branches indicates the distance


.

A short horizontal distance - small intensity


differences between the two replicates.

A short vertical distance - the both replicates are


similar.
• Protein kinase mutants of
KO-58 (cmk1) KO-61(wis1) KO-67(mek1)
have correlated expression profiles with same
biological process and /or co-regulated.

• Up-regulation (Induction) and Down regulation


(Suppression) of different PK genes involved in the
cell division cycle signaling pathway of PK mutants
strains helps us to understand the regulation of the
cdc genes in Wild type S. pombe.

• Comparison of Genomic expression programs within


and across different yeast can illuminate and explain
the physiological process within each organisms and
can reveal evolutionary differences in the regulation
of genomic expression programs.
Thus micro array helps
- to assay gene expression within a sample or to compare
gene expression in two different cell types.
- to detect mutation or polymorphism in a gene sequence
- to understand the growth and development
- to explore the underlying genetic causes of many human
diseases, especially cancer.
[cancer results from defects in fundamental cell regulatory
mechanisms, it is a disease that ultimately has to be understood at
the molecular and cellular levels.]

• APPLICATION
- tissue/cell phenotype analysis by multiple gene expression assay,
- identification of pathogens,
- genome-wide mutation survey
- diagnosis of genotypic polymorphisms and mutations.
- drug discovery,
- developmental biology
- molecular analysis of various diseases.