Studies On Protein Kinase Signalling Pathway Involved In

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.

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Schizosaccharomyces pombefission yeast.

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Pombe is derived from the Swahili word for beer.

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Isolated in 1893 by Lindner from millet beer.

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The sixth model eukaryotic organism whose genome has been completely sequenced.

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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 kinase activity. cyclins • 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

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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 KO 42-pck1; KO 67-mek1;

Each column - a sample.

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 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.