Studies on Protein Kinase Signalling Pathway Involved In

8/14/2019 Studies on Protein Kinase Signalling Pathway Involved In

1/24

STUDIES ON PROTEIN KINASESTUDIES ON PROTEIN KINASE

SIGNALLING PATHWAY INVOLVED INSIGNALLING PATHWAY INVOLVED IN

SCHIZOSACCHAROMYCES POMBESCHIZOSACCHAROMYCES POMBE

GENOME INSTITUTE OF SINGAPORE

M.SHANMUGA PRIYAA

M.Sc BIOTECHNOLOGY


2/24


3/24

Comparative study, on the expression of proteinComparative study, on the expression of protein

kinase genes pertaining to the signaling pathway ofkinase genes pertaining to the signaling pathway of

the cell division cycle (cdc), ofthe cell division cycle (cdc), ofS.pombeS.pombe in both thein both the

wild and mutant strains is being investigated usingwild and mutant strains is being investigated using

micro array technology.micro array technology.


4/24


5/24

Schizosaccharomyces pombe-fission yeast.

Pombe is derived from theSwahiliSwahiliword for beer.

Isolated in 1893 by Lindner frommilletbeerbeer..

The sixth model eukaryoticorganism whose genomegenome has beencompletely sequencedsequenced..

S.pombe is,

an unicellulareukaryoteeukaryote,,rod-shaped

2 - 3 m in diameter

7-14 m in length

WILD MUTANTWILD MUTANT
http://en.wikipedia.org/wiki/Swahilihttp://en.wikipedia.org/wiki/Swahilihttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Swahili


6/24

Key players in the progression of the cell cycle are a group ofkinases.

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.


7/24

KINASES

Binds to cyclinscyclins-regulate kinase activity.

Inactive kinases-cdkscdks

Add phosphate group

Phosphorylation of proteins at different stages of the cell cycle is

necessary for the cycle to complete.


8/24

GENE EXPRESSION:

ComplexComplex pattern of expression

Determines the characteristicscharacteristics of a cell and its rolerole in the organism.

Abnormal pattern- cancercancer..

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 ofDNA chipsDNA chips using known DNAs.2.Generation of a hybridization solution containing a mixture of

fluorescently labeled cDNAslabeled cDNAs (mutant type).

3.IncubationIncubation of the hybridization mixture containing fluorescently labeled cDNAs with

DNA chip.

4.Detection of bound cDNAs using llaserasertechnologytechnology and storing data in a computer.

5.Analyzing and interpreting the data using computationalcomputational methods.methods.


9/24


10/24

Cell cultureCell culture

Total RNA extractionTotal RNA extraction

Reverse Transcriptional reactionReverse Transcriptional reaction

CDNA probe preparationCDNA probe preparation

Coupling CYS dyesCoupling CYS dyes


11/24

Preparation of Micro

array slides.

Spotting of arrays


12/24

Hybridization

Scanning

Yellow =equal expression


13/24


14/24

The image analysis - 2 final intensity measurements for the red and greenThe image analysis - 2 final intensity measurements for the red and green

are obtained for every single spot.are obtained for every single spot.

GreenGreen represents - the expression of the control DNA (wildwild),

RedRed represents - the expression of the Sample DNA (referencereference),

yellowyellow represents - the expression of both the controlcontrol and samplesample

DNA.

BlackBlack represents - the expression of the neither the control norsample.

SCANNED SLIDESCANNED SLIDE


15/24

CLUSTERED IMAGE PRODUCED USING GENE PIX PRO4.1CLUSTERED IMAGE PRODUCED USING GENE PIX PRO4.1

KO 42-pck1; KO 49-cdc2; KO 51-chk1; KO 55-fin1; KO 58-cmk1; KO 59-orb6;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 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;KO 67-mek1; KO 75-kin1; KO 76-cdc7; KO 77-wee 1; KO 81-win 1;

Each rowrow -- a gene Each columncolumn - a sample.


16/24

The pattern of expression of Protein kinase mutant

strain

Orb6 (KO-59 A &B)

Each rowrow -- a gene

GreenGreen color - a particular

gene in the mutant is suppressedsuppressed

(down regulated).(down regulated).

RedRed color- a particular gene

in the mutant is induced (upinduced (up

regulated).regulated).

GREYGREY color - the missing data.

BLACKBLACK color - no expression


17/24

The pattern of expression of Protein kinase mutant strain

Plo1 (KO-60 A &B)

GreenGreen color - a particular gene in

the mutant is suppressed.suppressed.

RedRed color - a particular gene

in the mutant is induced.induced.




18/24


Wis1 (KO-61 A &B)








19/24


Wee1 (KO-77 A &B)







T i f P t i ki t t


20/24

Tree view of Protein kinase mutants

A short horizontal distanceA short horizontal distance - small intensity

differences between the two replicates.

A short vertical distance - the both replicates aresimilar.

The length of the branches indicates the distanceThe length of the branches indicates the distance

..


21/24

Protein kinase mutants of


22/24

Protein kinase mutants of

KO-58 (cmk1) KO-61(wis1) KO-67(mek1)

have correlated expression profiles with samebiological process and /orco-regulated.

Up-regulation (Induction) and Down regulation(Suppression) of different PK genes involved in thecell division cycle signaling pathway of PK mutants

strains helps us to understand the regulation of thecdc genes in Wild type S. pombe.

Comparison of Genomic expression programs within

and across different yeast can illuminate and explainthe physiological process within each organisms andcan reveal evolutionary differences in the regulationof genomic expression programs.

Thus micro array helps


23/24

APPLICATIONAPPLICATION

- tissue/cell phenotype analysis by multiple gene expression assay,

- identification ofpathogens,

- genome-wide mutation survey

- diagnosis of genotypic polymorphisms and mutations.

- drug discovery,

- developmental biology

- molecular analysis of various diseases.

Thus micro array helps

- to assay gene expression within a sample orto compare

gene expression in two different cell types.

- to detect mutation orpolymorphism in a gene sequence

- to understand the growth and development

- to explore the underlying genetic causes ofmany humandiseases, 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.]


24/24

Documents

Studies on Protein Kinase Signalling Pathway Involved In