Eukaryotic Cell Cycle

8/4/2019 Eukaryotic Cell Cycle

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Eukaryotic

Cell CycleFor M.Sc. Final Year (4th Semester) + (1stSemester)


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Eukaryotic Cell Cycle=Life cycle of a Cell

Consists of 4 co-ordinated processes:

1. Cell growth

2. DNA Replication

3. Distribution duplicated cells to daughter cells

4. Cell Division

Birth Growth Reproduction Death

Interphase


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Cell Cycle

Middle of 19th Century

Cell growth & Division Single cellbacteria to Multicellular organ.

Cells duplicates its contents Divide

Cell cycle


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Each individual must manufacture millions of cells

every second to survive.

To Replace dead cells Apoptosis.

To passing of genetic information.

To receive accurately the replicated

chromosome.

To receive copy of entire genome.


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Cell Cycle

Somatic Cell Division - Overview

Interphase 95% of cell cycle

Organelle duplication, DNA replication,

Growth


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G1 Phase

Metabolically active

Organelle duplication, but no DNA replication

Duration variable short in embryonic and

cancer cells

Prepares for S phase

Cells that remain in G1 for a long time = G0

(permanent tissues, such as neural tissue)


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G2 Phase

Growth continues

Enzymes and proteins synthesized for

cell division Determining Cell Stage

Cells at different stages of the cell cycle

can also be distinguished by their DNAcontent


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S Phase

Committed to cell division once this starts

DNA and centrosome replication

Semi-conservative replication of DNA:two identical daughter genomes


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Blood Lymphocyte/Fibroblasts/ Epithelial cells

Human Cell Cycle 24 hrs.

Interphase 23 hrs.

Two basic parts

Mitosis 1 hr.

(Cytokinesis)

Go

Interphase = 23 hrs

M G1 S G2 1hr

(Gap.1) Synthesis (Gap 2)

11 hrs 8 hrs 4 hrs

Metabolically active Growth Protein

growth synthesis

Ana

meta

Pro

M Telo


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Exceptions:

1. Budding yeasts 4 phases in 90 minutes.

2. Early Embryo cells 30 min only or less.

(after fertilization of eggs) No growth takes place but separately synthesize

DNA & divide.

No G1 and G2 stage.

3. No division at all Nerve cells.

4. Only occasionally divide skin Fibroblast/Liver

on demand or for

repair/replacement.


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Cell cycle Regulation in yeast

A comparison of the cell cycles of fission yeasts and budding yeasts(A) The fission yeast has a typical eucaryotic cell cycle with G1, S, G2, and M phases. In contrast with what happens in higher eucaryotic cells, however,

the nuclear envelope of the yeast cell does not break down during M phase. The microtubules of the mitotic spindle (light green) form inside the nucleusand are attached to spindle pole bodies (dark green) at its periphery. The cell divides by forming a partition (known as the cell plate) and splitting in two.

The condensed mitotic chromosomes (red) are readily visible in fission yeast, but are less easily seen in budding yeasts. (B) The budding yeast hasnormal G1 and S phases but does not have a normal G2 phase. Instead, a microtubule-based spindle begins to form inside the nucleus early in the cycle,

during S phase. In contrast with a f ission yeast cell, the cell divides by budding. As in fission yeasts, but in contrast with higher eucaryotic cells, thenuclear envelope remains intact during mitosis, and the spindle forms within the nucleus.


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Regulation of the cell cycle of budding

yeast

The cell cycle ofSaccharomyces cerevisiae is regulated primarily at a point in late G1 called START.

Passage through START is controlled by the availability of nutrients, mating factors, and cell size.

Note that these yeasts divide by budding. Buds form just after START and continue growing until they

separate from the mother cell after mitosis. The daughter cell formed from the bud is smaller than the

mother cell and therefore requires more time to grow during the G1 phase of the next cell cycle.

Although G1 and S phases occur normally, the mitotic spindle begins to form during S phase, so thecell cycle of budding yeast lacks a distinct G2 phase.


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The morphology of budding yeast cells

arrested by a cdcmutation

(A) In a normal population of proliferating yeast cells, buds vary in size according to the cell-cycle

stage. (B) In a cdc15mutant grown at the restrictive temperature, cells complete anaphase but

cannot complete the exit from mitosis and cytokinesis. As a result, they arrest uniformly with the

large buds, which are characteristic of late M phase


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The behavior of a temperature-sensitive cdc

mutant

(A) At the permissive (low) temperature, the cells divide normally and are found in all phases of the cycle (the phase of the cellis indicated by its color). (B) On warming to the restrictive (high) temperature, at which the mutant gene product functionsabnormally, the mutant cells continue to progress through the cycle until they come to the specific step that they are unable to

complete (initiation of S phase, in this example). Because the cdcmutants still continue to grow, they become abnormallylarge. By contrast, non-cdcmutants, if deficient in a process that is necessary throughout the cycle for biosynthesis and

growth (such as ATP production), halt haphazardly at any stage of the cycledepending on when their biochemical reservesrun out.


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In Animal Cells

The Decision point at late G1 is called:

Extracellular Growth Restriction Point = START

Factors signaling.

If growth factor is not available cells enters Go, the quiescentstage of cell cycle.

Go stage metabolically active

cease growth & proliferation

Less protein synthesis. Wound

Blood Platelets

Skin fibroblasts activated to grow if there is wound PDGF ClottingSignals proliferation

of fibroblasts/skin cells

near injury


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Cell Cycle in Schizosaccharomyces pombe

Cell cycle control at G2 instead of G1

G2 M

Cell size nutrient etc.

monitored

In mammals/animals Oocytes remain at G2 for years/

decades. If not stimulated

by hormones


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Cells produces Proteins that forms Cell

cycle control system that control

progression of cell cycle.

Central role of controlling no of cells in the

body.


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In HumanG1 S G2 M

Schizosaccharomyces pombe (Fission yeast)

Yeast system

Saccharomyces cerevisiae (Budding yeast)

Mutation in cell division cycle genes (cdc genes)


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Cell Cycle

= Life cycle of a cell=

Birth Growth Reproduction Death

Divided into different Phases G1 S G2 M Go

9-12 hr G1 = growth & preparation or DNA duplication 16 steps Synthesize RNAs & Proteins10-12 hr S = Synthesis of genetic material (DNA)

4-5 hr G2 = growth & preparation for cell division.

1/2 -1 hr M = Mitosis

Cell cycle controls Cell Differentiation

brain cells

Stem cells into specialized cells Liver cells

skin cells

In adult person bone marrow stem cells differentiate to blood cells


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Driving forces are Specific Proteins present in the cell (Kinases & Cyclins)

Controlled by Cell Division Cycle genes = cdc genes

Cdc genes instruct the cells to produce required Kinase to operate cell cycle.

Cell Cycle Kinases (protein enzymes) synthesized by cdc 2 gene Phosphorylation.

Human DNA (genome) 3 billion bp 30,000 genes = 23 pairs of chromosomes


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Cell cycle:

An orderly sequence of macromolecular events

that allow duplication of cellular contents and cell

division leading to production of two daughter cellseach containing identical copy of genetic materials

of the parental cell.


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Cell division and cell cycle occur in proper orderand with high fidelity.

Regulation of cell cycle is critical for normaldevelopment and loss of control leads toCANCER.

Cell replication is primarily controlled by regulatingtiming of nuclear DNA replication and mitosis.

The master molecules that control cell cycleevents are a number ofHeterodimeric ProteinKinases.


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Regulatory submit Cyclins

Catalytic submit Cyclin dependent Kinases (cdks)

(regulate through phosphorylation)

DNA 2 no levels

Chromosomes Protein (nuclesome) Histones & Chromosomal

proteins

RNA

Interphase = (G1 s G2)

Mitosis (M)

Prophase Metaphase Anaphase Telophase


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Human cell cycle 24 hrs

Exceptions:

Some cells (Post mitotic) differentiated cells like Nerve cells/Retinal/Eye lens

cells.

Exit Cell Cycle enter in Go phase remain longtime without dividingQuiescent Stage.

..

Anaphase Promoting ComplexCyclosome

PRO Meta Anaphase Telaphase


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Cell cycle control Check Points = Control system blocksprogression through these check

points if it detects problems

inside or out side thecells.

1. Restriction point/Start Late G1

(Commits cell cycle entry) G1/s

2. G2/M check point Early mitotic events phosphorylation

chromosome condensation, spindle assembly etc.

3. Metaphase to Anaphase Transition.

(APC/C protein) Anaphase promoting complex cyclosome

(Stimulates sister chromatid separation) leads to completion of mitosis

& cytokines)


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Cell-Cycle Checkpoints

G1 checkpoint In yeast, called start

In animal cells, called restriction point

G2 checkpoint Located at boundary between G2 and M phase

Proper completion of DNA synthesis required before

cell can initiate mitosis

Spindle Assembly Checkpoint Boundary between metaphase and anaphase

All chromosomes must be properly attached to the spindle


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Cell Cycle Control System = central component in cdks

Cdks are regulated by Cyclins.

Without cyclins cdks do not function/inactive.

When Cyclin forms the complex with cdk, protin kinase is activated &

trigger cell cycle events.

4 classes of cyclins each defined by the stage of the cell cycle at

which they bind to cdks & function.

All eukaryotic cells require 3 of these 4 cyclins.

Two key components of the cell-cycle control system


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1. G1/s cyclins activates cdks at G1 Startcommit entry to cell cycle. (G1/S-cdk) complex.

2. S Cyclins bind cdks soon after progressionthrough Start. (S cdk) complex.

3. M Cyclins activates cdks that stimulates entry into

Mitosis at G2/M check points (M cdk)complex.

4. G1 cyclins Govern the activities of the G1/Scyclins & controls progression through start in G1.

In vertebrates:

4 Cdks G1 cdk, G1/S cdk, S cdk and M - cdk


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3 D pictures of Cdk & cyclin proteins

In absence of Cyclin active site of Cdk is obscured by a slab protein.

Binding of Cyclin partial activation of cdk enzyme.

Phosphorylation by Cdk activating

Kinase (CAK)

(a.a. at cdk active sites)

Causes Conformational change

Activate cdk Phosphorylate target protein

Induce Cell Cycle regulation.


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Regulation of Cyclin Cdk complex Activation(by 3 D pictures of Cdk 2)

A. In inactive state without cyclin bound, the active site is blocked by T loop

protein.

B. Binding of cyclin causes T loop to move out of active site causing partial

activation of Cdk 2.

C. Phosphorylation of Cdk 2 by Cdk activating kinase (CAK) at threorineresidue in T loop activate the enzyme fully and allow the enzyme to bind its

protein substrates.

Cdk activity can be suppressed by inhibitors

Phosphorylation of Cyclin Cdk complex by a protein kinase Wee 1inhibits Cdk activity.

Dephophorylation of Cdk sites by phosphatase known as cdc 25 incresesCdk activity.


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Cell Cycle Control by step wise Proteolysis

Activation of specific cyclin cdk complexes drives progression through START & G2/M check points.

Progression through Metaphase to Anaphase transition is triggered by protein destruction Lead to finalstages of Cell Division.

APC/C (Anaphase promoting complex/cyclosome ubiquitin by gase enzyme)

Ubiquitylation &

destruction of

Securin protects proteins linkages thathold sister chromatids together.

Separates sister chromatides

(unleashes anaphase)

Destroy S and M cyclins.

Dephosphrylation of proteins

& Cdk targets.

Cytokinesis Cell Division -


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APC/C remains active in G1 Cdk remains inactive

Late G1 G/S Cdk gets activated

APC/C turn off.

Allow Cyclin accumulation

Start Next cell cycle


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Cyclin cdk complex

1. G1 cdk cdk4 + cdk6 + Cyclin D

2. G1/S cdk cdk2 + Cyclin E

3. S cdk cdk2 + cdk1 + Cyclin A

4. M cdk cdk1 + Cyclin B


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Cell Cycle Control System has a central role in

regulating cell numbers in the tissues of the body.

If this system malfunctions leads to excessive cell

divisions cancer.

With genome duplication cells need to duplicate also otherorgancells and macromolecules & incerese cell mass.

Segrate the all copies precisely into two genetically

identical cells.


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How to study Cell Cycle?

Human Fibroblast 25 40 divisions Replicative cellsenescence

Cells in culture

Transfetion mutation

Immortalized cell lines used for cell cycle study

genetically homogeneous cells.


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Direct visualization of growing cells at differentstages of cell cycle as cells rounded up in mitosis.

By using DNA binding fluorescence dyes.

Or antibodies staining. S-Phase can be studied by 3H thymidine & 5

BrdU (Bromodeoxyuridine)

Visualized by staining with antiBrd U

antibodies/fluorescence dyes.

How to study Cell Cycle?.........


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Biochemical, genetic and molecular

biology techniques have been employed to

studying various aspects of cell cycle.

Cell replication is primarily controlled by

timing of nuclear DNA replication &

Mitosis.


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I

II

III

I

II


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In each cell Amount of fluorescence = amount of DNA Present

First 1. unreplicated complement of DNA G1

Second 2. Fully replicated complement of DNA G2/M phase

(Twice G1 DNA content)

Third 3. Intermediate amount of DNA S - Phase

By measuring DNA content 2n/2c value by 4C in G2 phase by Flowcytometer

(FACS) G1 (Fluorescence activated cell sorting)

How to study Cell Cycle?.........


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This graph shows typical results obtained for a

proliferating cell population when the DNAcontent of its individual cells is determined in a

flow cytometer. (A flow cytometer, also called a

fluorescence-activated cell sorter, or FACS, can

also be used to sort cells according to their

fluorescence. The cells analyzed here were

stained with a dye that becomes fluorescent

when it binds to DNA, so that the amount of fluorescence is directly proportional to the

amount of DNA in each cell. The cells fall into

three categories: those that have an

unreplicated complement of DNA and are

therefore in G1 phase, those that have a fully

replicated complement of DNA (twice the G1

DNA content) and are in G2 or M phase, andthose that have an intermediate amount of DNA

and are in S phase. The distribution of cells in

the case illustrated indicates that there are

greater numbers of cells in G1 phase than in G2

+ M phase, showing that G1 is longer than G2 +

M in this population.

Analysis of DNA content with a flow cytometer


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Dissection of Cell cycle using yeast mutations

Two yeast species 1. Fission Yeast = Schizosackharonyces pombe

used in cell cycle (used in producing beer)study (1% of gene) 2. Budding yeast = Saccharomyces cerevisae

(brewer & bakers)

Genes can be replaced, deleted or altered.

Proliferate in hyploid state

Easy to manupulate the gene

as it is one copy of genome. Temperature sensitive Mutation

Search mutation in yeast that Low Temperature High Temperature

inactivate genes encoding essentialcell cycle regulatory genes = cdc gene Normal Prolif Mutation

(No Mutation cdc) (cdc Mutation)

Yeast flow in different by seeing the

phases of cell cycle. buds/size of the bud


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Example: cdc15 mutant at high temperature complete upto anaphase but can notcomplete anaphase sepration and cytokinesis. The buds became large & remain late

M phase.

Cell cycle = Circulation Rhythm day night day cycle

Chronobiology Monthly cycle

Chronopharmacology Yearly cycle

(circa.)


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Regulation of the Cell

Cycle

Cell Cycle Lengths

Vary by cell type: Embryonic cells

Stem cells (e.g., blood cells and epithelial cells) Sperm cells

G1 prolonged in stable or permanent cells(called G0)

G1 rapid or non-existent in rapidly-dividing

cells


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Embryonic cells

Cell growth not part of cell cycle

All energy goes into DNA synthesis

So G1 lacking and G2 quite short Each round of division subdivides original

cytoplasm into smaller and smaller cells,

Until adult cell size is reached


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Cell Cycle Regulation

Protein Phosphorylation & Degradation of proteins control

passage through cell cycle.

1. Concentration ofCyclins the regulatory submits of heterodimeric

protein kinases (Cdks) catlytic submit that control cell cycle eventsincrease or decrease as cells progress through cell cycle.

2. CDKs have no kinase activity unless they are associated with cyclins.

3. Associated cyclin determines which proteins to be phosphorylated bythe cyclin-cdk complex.


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Current model for regulation of the eukaryotic cell cyclePassage through the cycle is controlled by G1, S-phase, and mitotic cyclin-dependent kinase complexes (CdkCs) highlighted in green. These are composed

of a regulatory cyclin subunit and a catalytic cyclin-dependent kinase subunit. Protein complexes (orange) in the Cdc34 pathway and APC pathwaypolyubiquitinate specific substrates including the S-phase inhibitor, anaphase inhibitor, and mitotic cyclins, marking these substrates for degradation

by proteasomes. These pathways thus drive the cycle in one direction because of the irreversibility ofproteindegradation. Proteolysis ofanaphase inhibitorsinactivates the protein complexes that connect sisterchromatids at metaphase (not shown), thereby initiating anaphase.


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Cell Cycle Regulation

3 major cyclin cdk complexes controls:

1. G1 cyclin cdk complex express first to stimulate replication at late G1

2. S cyclin cdk complex

3. Mitotic cyclin cdk complex

Activate Transcription factors

Promote

Transcription of Genes codes for

1. Enzymes for DNA synthesis2. Sphare cyclins

3. S phase cdks

S phase cyclin cdk complex is checked by inhibitors.


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At late G1 G1 cyclin cdk complex induce degradation of inhibitors by phosphorylation &

ubiquitination by SCF ubiquirtin lygase.

Activates/initates Phosphorylate regulatory sites Releases active

Replication of DNA of proteins DNA prereplication S phase Cyclin -

Complex cdk complexes

Prevents assembly Assembled at

Of pre replication complex Replication Origin

Because of this inhibition

Each chromosome is replicated once

So proper chromosome number is

Maintained in daughter cells.


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Once activated, S-phase Cyclin Cdk complexes

phosphorylates regulatory sites in Protein that form DNA

pre-replication complexes that assembled at DNA

Replication Origin during G1.

Phosphorylation of these proteins activates initiation of

replication and also prevents reassembly of pre

replication complex again.

This inhibition makes each chromosome to replicate once

during on cell cycle, thus maintains specific chromosome

number.

Mitotic cyclin cdk complexes are synthesized in S and

G2 phase but they are held in check by phosphorylation at

inhibitory sites till the DNA synthesis is complete.


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Once activated by dephosphorylation at

inhibitory sites, mitotic cyclin CDK complexes

phosphorylates multiple proteins that lead to

1. Chromosome condensation

2. Retraction of nuclear envelope

3. Assembly of mitotic spindle apparatus

4. Alignment of chromosomes at metaphase plate


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During mitosis APC (Anaphase Promoting Complex), a

multisubmint ubiquitin lygase polyubiquitinates key

regulatory proteins proteosomal degradation.

APC ubiquitinates Securin that inhibits degradation of

cross-linking protein between sister chromatids. But

degradation of Securin by APC is inhibited until the

kinetochores assembled at centrosomes of allchromosomes and attached spindles microtubules and

aligned at metaphase plate.

Following Securin degradation initiation of Anaphaseoccurs by freeing sister chromatids to seggregate to

opposite poles.


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At late Anaphase APC directs ubiquitination and proteosomal

degradation of mitotic cyclins. But this inhibited until the

chromosomes have reached proper location in dividing cell.

It leads to:

1. Inactivation of protein kinase activity of mitotic CDKs

2. Active protein phosphasases remove phosphates from specificproteins.

3. Separated chromosomes start decondensing.

4. Nuclear envelope reforms around daughter nuclei

5. Golgi apparatus reassembles at Telophase

6. Cytoplasm devides at Cytokinesis resulting in two daughter cells.


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During the early G1 of next cell cycle, phosphatasesdephosphorylates proteins that Pre-Replication

complex.

Because of dephosphorylation at G1, a new pre-

replication complexex are able to reassemble at

replication origins in preparation for the next S phase.

Phosphorylation of APC by G1 Cyclin CDK complexes

at late G1 inactivates it allowing accumulation of S-

phase and mitotic cyclins during ensuing cycle.


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Passage through three critical cell cycle transitions

1. G1 S phase

2. Metaphase to Anaphase

3. Anaphase to Telophase & Cytokinesis

This is an irreversible process because the above

transitions are triggered by the regulated degradation

of proteins.

Therefore cells are forced to traverse the cell cycle in

one direction only.


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The inhibition of a cyclin-Cdk complex by CKI. Three-dimensional structure of

the human Cyclin A-Cdk 2 complex bound to the CKI p27, as determined by X-ray

crystallography. The p27 binds to both the cyclin and Cdk in the complex distorting

the active site of the Cdk.


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(A) Control of proteolysis by APC/C

The control of proteolysis by APC/C and SCF during the cell cycle. (A) The

APC?C is activated in mitosis by association with the activating subunit Cdc20,

which recognizes specific amino acid sequences on M-cyclin and other target

proteins. With the help of two additional proteins called E1 and E2, the APC/C

transfers multiple ubiquitin molecules onto the target protein. The polyubiquitylated

target is then recognized and degraded in a proteasome.


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The activity of the ubiquitin ligase SCF depends on substrate-binding subunits called

F-box proteins, of which there are many different types. The phosphorylation of a

target protein, such as the CKI shown, allows the target to be recognized by a

specific F-box subunit.

(B) Control of proteolysis by SCF


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What is SCF?

SCF complexes consists of three submit Proteins: SKP 1,

Cul 1 and Rbx 1. This alongwith a variable component, F

box Protein that binds to SKP 1 through F box motif that

recognizes substrates.

F box protein in combination with Skp 1, Cul 1 and Rbx 1

as well as E2 proteins provides basis for multiple substrate

specific ubiquitination pathways.

S f h M j C ll l R l P i


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Summary of the Major Cell-cycle Regulatory Proteins


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Cell Differention

Ability to give rise to hetrogeneous progeny of cells which progressively

diversify and specialize and constantly replenish short lived tissues.

Mother Cells

Undifferentied STEM CELLS No function

(Self renewal capacity)

Differentiated to

Skin cells Nerve cells Blood cells Muscle cells Immune

cells


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Development of organs & tissues in multicellular organisms depends on specific pattern of mitotic celldivision.

A series of such cell division in a family tree called celllineage which can trace the birth order, developmentalpotential and differentiation to specialized cell types.

Cell lineages are controlled by both internal andexternal factors. cells cell signals and environmentalfactors.

A cell lineage begins with Stem Cells the name derivesfrom plant stem. It ultimately form terminallydifferentiating cells (Nurons, skin, liver) which is airreversible (??) process.


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Terminally differentiating cells generally donot divide but

survive to do all functions for particular length of time

and then die.

Many linages contain intermediate cells called

Precursor/Progenitor cells. Which can form different

types of differentiated cells.

Precursor cells once created, produce various

Transcription factors which activate or repress many

genes that direct/control differentiation process.

Programmed cell death is essential for maintenance of

all tissues and hence a balance is maintained in birth,

growth and death of cells in the body.


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Symmetrical division produce duplicates of patental cells

in all respects.

But daughter cells produced by a symmetric cell division

differ in shape, size composition and genes of different

state of activity.

Stem cells divide a symmetrically to generate a copy of

itself and a derivative stem cell that has more restrictive

property like growing for limited period.


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Needs replacement of aged, injured or diseased cells.

Blood cell turn over = 108 -109 cells/hr.

Epithelial cells in intestine = 3 5 days.

Skin cells turnover = 3 4 weeks.


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8 cell stage mice cell 8 cells are totipotent.

Neurons (transmitting) Propagating

electical signalsParticular Progenitor Cells divides

Gial cells Provides electrical insulation

Stem Cells

Neurons Neurotransmitter Dopamine

Dopamine producing neurons Transplant embroyonic neurons in

No rejection by immune system Parkinson patient.

Diff ti ti St


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Differentiation: Stem

cells

so fertilization of the eggtakes place in the oviduct

the fertilizes zygote travelsto the uterus for implantation

along the way the zygotebegins to divide (mitosis)

2-cell, 4-cell, 8-cell

embryonic stages etc. the embryo reaches a stage

called the morula = ball ofsmall cells (embryo has notenlargened)

by the end of the first weekthe second embryonic stage

the blastocyst- forms

Differentiation: Embryonic Stem


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Differentiation: Embryonic Stem

cells

the ES cells are said to be totipotent have the ability to specialize or differentiateinto ALL cells of the embryo

the blastocyst then begins a process of differentiation and these ES cells formpopulations of stem cells with more restricted potentials

the ES cells first differentiate into two layers called the embryonic disc divides theblastocyst cavity into an amniotic cavityand a yolk sac(primitive hematopoieticorgan)

these two layers then continue to differentiate into the three germ layers of theembyro ectoderm, mesoderm and endoderm

the formation of these germ layers marks the gastrula embryonic stage

the blastocyst is a hollowball of cells containing an

outer rings of progenitorcells = trophoblastand aninner mass of cells at oneend of the embryo = innercell mass

it is these ICM cells that arethe source for thederivation ofembryonicstem (ES) cells


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Germ Layers

the ectoderm, mesoderm and endoderm are thought tobe made up of stem cells with a more restrictedphenotype when compared to ES cells BUT still capableof forming multiple cell types within that lineage

e.g.pluripotent stem cells interactions between signaling molecules produced by

these germ layers and with the developing ECM aroundthese tissues results in specific developmental events =

patterning

patterning requires the exposure of cells to a successionof signals and subsequent activation of their associatedpathways


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Molecular Mechanism of Differentiation to Specialized cells.

Cells of higher organisms switch genes on or off in response to

environmental changes-evolve specialized ways to form differential

cell types.

Once a cell committed to differentiate into specific cell types, cells

maintains this phenomena through many generations i.e. cells

remember the changes in gene expression. This is called cell

Memory.

Cell memory is an essential prerequisite for creation of differential

tissues and stably differentiated cell types.

In eukaryotes and bacteria other changes in gene expression are

transient. For example, Tryptophan Gene.

DNA t di t h i ti i b t i


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DNA rearrangement mediate phase variation in bacteria.

Cell differentiation in higher eukaryotes generally occur without any

change in DNA sequence.

In Prokaryotes gene regulation is achieved by DNA rearrangements that

activate or inactivate gene expression.

During DNA replication cycles altered gene sequence will be inherited by

all progeny of cells.

But some rearrangements (changes) are found to be reversible so that

individual can switch back to the original DNA configuration.

Salmonella is known to show such interesting phenomenon ofPhase

Variation.

S h d f diff ti ti h k t t


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Such mode of differentiation has no known counterpart

in high eukaryotes.

This type of phase variation has significant impact on

animals because disease causing bacteria use it to

evade detection by immune system of the host.

Salmonella gene expression is brought about byoccasional inversion of specific 1000 bp DNA. This

change alters the expression of cell surface protein

Flagellin which is encoded by two different genes.

A site-specific recombinant enzyme catalyses the

inversion leading to changes the orientation of

Promoterlocated within the invested segment.

If The promoter in one orientation the bacteria can


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If The promoter in one orientation, the bacteria cansynthesize one type ofFlagellin while Promoter in otherorientation, they synthesize the other type of Flagellin

protein.

Inversions occurs only rarely and that is why bacteriagenerally have one or the other type ofFlagellin.

This type of inversion by phase variation protectsbacterial population against the immune response of thehost.

If the host makes antibodies against one type Flagellin,some bacteria which have synthesized other flagellin bygene inversion will be able to survive and multiply.


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Wild type of bacteria is exhibit phase variation whilelaboratory bacterial strains lose this character overtime and not all involve DNA inversion.

Exceptions

Nisseriia gonorrhoeae avoids immune attack by heritable

changes in its surface properties that arises form geneconversion instead of gene inversion.

Transfer DNA sequences from a library of silent genecassettes to the site of the genome where genes are

expressed.

It creates many variants of major bacterial surface protein.

Molecular Genetic Mechanisms that create specialised cells


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Molecular Genetic Mechanisms that create specialised cells.

Cell Memory: When cells are commited to differentiate to a specific cell type, cellsremembers the changes in gene expression for several generations.

Other changes in gene expression in eukaryotes & bacteria are transient.

+Tryptophan

Example: Tryptophan gene switched off

in Bacteria

(Sal monella) Remove Trisptophan

Gene is switched on.

Bacterial cells will not have anymemory that cells were exposed toTryptophan

DNA reaggangements mediate phase Variation.

Cell differentiation occur in

higher eukaryotes without

detectable change in DNA

sequence.

B t i


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Bacteria

Differention Mechanism

Yeast

Phase Variation Salmonella bacteria.

It has no equivalent in higher eukaryots.

Disease causing bacteria use it to evade detection by hosts immune system.

It occurs to inversion of 1000 bp, DNA

Site specific Leads to change in expression of cell surface protein = Flagellin

Recombination 2 genes are responsible.

enzyme

Change orientation of Protaoter synthesize one type of

Flagellin

Other orientation Other Flagellin type is synthesized.

It protects bacteria because when the host makes antibodies against one type of


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Flagellin, there bacteria will still survive & multiply because of other Glagellin protein

being synthesized by other bacteria.

Bacteria from wild often show Phase Variation.

Laboratory strains lose this trait.

Not all bacteria involve inversion.

For example, Neisseria genorrhoeae.

avoids immune attack by heritable

change in surface properties cared by gene conversion

Transfer DNA sequences for Library of Silent gene

cassettes to the site of gene expression.

Create many variants of bacterial surface protein.

S it hi G i b DNA I i i B t i


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Switching Gene expression by DNA Invension in Bacteria

Switching gene expression by DNA inversion in bacteria. Alternatingtranscription of two flagellin genes in a Salmonella bacterium is caused by a simple site-specific

recombination event that inverts a small DNA segment containing a promoter. (A) In oneorientation, the promoter activates transcription of the H2 flagellin gene as well as that of a

repressor protein that blocks the expression of the H1 flagellin gene. (B) When the promoter is

inverted, it no longer turns on H2or the repressor, and the H1 gene, which is thereby released

from repression, is expressed instead. The recombination mechanism is activated only rarely

(About once in every 105 cell divisions).

Therefore, the production of one or other flagellin tends to be faithfully inherited in each clone

of cells.


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Studies with yeast mutants

Yeast carrying mutation in the Sterile 12 gene.(STE 12)

Cannot respond to this pheromone.

Do not mate.

STE 12 a transcription factor that binds to DNA at pheromone

response element (PRE) present in a or cells Upstream

Regulatory Sequence (URS).

Binding of mating factors to cell surface receptors induces a cascade of

signaling events resulting in phosphorylation of various proteins including

STE 12 which in turn stimulate transcription.

Gene Regulatory proteins determine cell type in yeast


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Gene Regulatory proteins determine cell type in yeast.

Saccharomyces cerevisiae, a single cell eukaryote which can

exist in 3 distinct forms:

1. Haploid a type

2. Haploid type

3. Haploid a/ type

Diploid cells form by mating of two hyploid cells that differ in mating type (sex)(i.e. and a) and fuse.

These two mating types ( & a) produce specific diffusible signalling molecule

(mating factor) and cell surface repressor protein.

These molecules help them to be recognized for mating and fusion.

Diploid cells called /a are distinct from parental cells in the sense that they are

unable to mate but can sporulate (when seen out of food) to form haploid cell

by meiosis.

Changing pattern of gene expression establishes the three


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Changing pattern of gene expression establishes the three

cell types.

The mating type of haploid cell is determined by a mating

type Mat locus.

Mat locus in a encodes a single gene regulatory protein Mat

a1 and in types two proteins Mat1 and Mat2.

Mat a1 protein has no effect in the a type haploid cells which

has produced this but important in Diploid cell.

In contrast, Mat2 protein acts in cell as Transcriptional

Repressorthat turns ofa specific genes.

Mat1 protein acts as a Transcriptional Acti ator that


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Mat1 protein acts as a Transcriptional Activatorthat

turns on the specific genes.

Once cells of two mating types have fused, the

combination of Mat a1 and Mat2 regulatory proteins

generates a completely new pattern of gene

expression. Unlike what was in potent cells.

This is one of the first study to show combinational gene

control in an eukaryotic system.

1 3 ll t ifi t i ti f t 1 2 1) d d b MAT


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1. 3 cell type - specific transcription factors 1, 2, a1) encoded by MATLocus and act in combination with general transcription factor MCM-1-mediate cell type specific gene expression.

asg = a specific gene/mRNAsg = specific gene/mRNA

hsg = Haploid specific gene/mRNA

2. Thus the actions of these three transcription factors can set the yeastcell on specific differentiation pathway cell minuting in a particular celltype.

3. Microarray study established expression and repression of many genesthat control cell characteristics.

4. Activity of MCM-1 is determined by its association with 1 and 2transcription factors.

5. As a combinatorial action, MCM 1 promotes transcription of specificgenes and repress a specific of genes in cells.

Control of cell type in yeasts Three


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Control of cell type in yeasts. Threegene regulatory proteins (Max1, Max2 and Mata

1) produced by the Mat locus determine yeast cell

type. Different sets of genes are transcribed in

haploid cells of type a, in haploid cells of type ,

and in diploid cells (type a/). The haploid cellsexpress a set of haploid-specific genes (hSG) and

either a set of -specific genes (SG) or a set of a-

specific genes (aSG). The diploid cells express

none of these genes. The Mat regulatory proteins

control many target genes in each type of cell by

binding in various combinations, to specific

regulatory sequences upstream of these genes.

Note that the Max1 protein is a gene activator protein, whereas the Max2 protein is a gene

repressor protein. Both work in combination with a

gene regulatory protein called Mcm 1 that is present

in all three cell types, In the diploid cell type, Max2

and Mata 1 from a heterodimer (shown in detail in

Figure) that turns off a set of genes (including the

gene encoding the Max1 activator protein)

different from that turned off by the Max2 andMcm1 proteins. This relatively simple system of

gene regulatory proteins is an example of

combinatorial control of gene expression.

T i ti f t d d t th M t 1 t i t ith M 1 t i t


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Transcription factors encoded at the Mat 1 ccus act in concert with Mcm1 protein to

specify cell type.

Each of 3 S cervrisiae cell types expresses a unique set of regulatory genes that

are responsible all the difference in three cell types of yeast.

Three cell type specific transcription factors, (1, 2 and a1) encoded at Mat locus

in combination with general transcription factor MCM1. (expressed in all three cells)

mediate cell type specific gene expression in yeast.

Haploid cells express a set of haploid specific genes hsg. a specific gene = asg.

specific gene = sg.

Diploid cells do not express any of these genes.

The Mat regulatory proteins (Mata Mata 1 & Mat 2) controls many


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The Mat regulatory proteins (Mata, Mata 1 & Mat 2) controls many

target genes in each type of cell by binding in various combinations to

specific upstream regulatory sequences (URS).

Mat 1 = Gene Activator protein

Mat 2 = Gene Repressor protein.

Both work in association with regulatory protein MCM-1 presents all 3

cell types.

In diploid cell type, Mat 2 & Mat 1 forms a heterotimer that turns off a

set of genes, (including gene for Mat 1) different from that turned offby Mcm 1 & Mat 2 proteins.

This is combinational control of gene expression.


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MCM 1 First member of MADS family of transcription factor (axrormym of 4 factors of

the family)

(Mini Chromosome Maintenance gene 1)

1. MCM 1 from budding yeast.

2. Agamous from Arabidopsis thaliana.

3. Deficiens from Snagdragon

4. SRF from Human

Role of MCM1 in a and yeast cells.


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Role of MCM1 in a and yeast cells.

MCM1 binds as a dimer to the P site in -specific and a-specific upstream

regulatory sequences (URSs), which control transcription of -specificgenes and a-specific genes, respectively.

a.) In a cells, MCM1 stimulates transcription ofa-specific genes MCM1 does

not bind efficiently to the P site in -specific URSs in the absence of 1

protein.

b.) In cells, the activity of MCM1 is modified by its association with 1 or

2. the 1-MCM1 complex stimulates transcription of -specific genes.

Whereas the 2-MCM1 complex blocks transcription ofa-specific genes.

The 2-MCM1 complex also is produced in diploid cells, where it has thesame blocking effect on transcription ofa-specific genes.


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Circadian clocks are based on Feedback loops in gene


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Circadian clocks are based on Feedback loops in gene.

Central role SCN Suprachiasmatic nucleus cells in

as time keep hypothalamus.on of mammals

Controls diurnal cycles of sleeping & walking

SCN cells neural cues from Retina dark light

Pineal gland release Melatonin

gives time signal of day and night.

SCN cells in culture behave show diurnalpattern of 24 hr. cycles


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Gene Regulatory Proteins determine cell type in yeast Single cell Eukaryote.


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g y yp y g y

Bakers yeast = Saccharomyces cerevisiae

1. Haploid

2. Haploid a

3. Diploid (a/)unable to mate but sporulate.

Mat locus Mating type & a

Mating factor

a type Mat locus Mat a1

type Mat locus Mat 1

Mat 2


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Gene regulatory Cell type/Yeast Set of genes controlled by Mat


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protein

1. Haploid a a

Mata

(No effect)

2.

(Haploid)

Mat 1 Mat 2

3. /a /a

(Diploid)

Mat 2 Mat a1

Eukaryotic Cell Cycle

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