Eukaryotic Cell Cycle

For M.Sc. Final Year (4th Semester) + (1st Semester)

Eukaryotic Cell Cycle=Life cycle of a Cell

Birth

Growth

Reproduction

Death

Consists of 4 co-ordinated processes: 1. 2. 3. 4.

Interphase Cell growth DNA Replication Distribution duplicated cells to daughter cells Cell Division

Cell Cycle
Middle of 19th Century Cell growth & Division Single cell bacteria to Multicellular organ. Cells duplicates its contents Divide Cell cycle

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.

Cell Cycle
Somatic Cell Division - Overview Interphase 95% of cell cycle Organelle duplication, DNA replication, Growth

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)

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 DNA content

S Phase
Committed to cell division once this starts DNA and centrosome replication Semi-conservative replication of DNA:
two identical daughter genomes

Blood Lymphocyte/Fibroblasts/ Epithelial cells

Human Cell Cycle 24 hrs. Interphase 23 hrs. Two basic parts Mitosis 1 hr. (Cytokinesis) Go
Pro

Interphase = 23 hrs M G1 S G2

Telo

1hr Ana

meta

(Gap.1) 11 hrs

Synthesis 8 hrs

(Gap 2) 4 hrs

Metabolically active growth

Growth Protein synthesis

Exceptions:
1. 2. Budding yeasts 4 phases in 90 minutes. 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. 4.

No division at all Only occasionally divide on demand or for repair/replacement.

Nerve cells. skin Fibroblast/Liver

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 nucleus and 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 has normal 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 fission yeast cell, the cell divides by budding. As in fission yeasts, but in contrast with higher eucaryotic cells, the nuclear envelope remains intact during mitosis, and the spindle forms within the nucleus.

Regulation of the cell cycle of budding yeast

The cell cycle of Saccharomyces 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 the cell cycle of budding yeast lacks a distinct G2 phase.

The morphology of budding yeast cells arrested by a cdc mutation

(A) In a normal population of proliferating yeast cells, buds vary in size according to the cell-cycle stage. (B) In a cdc15 mutant 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

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 cell is indicated by its color). (B) On warming to the restrictive (high) temperature, at which the mutant gene product functions abnormally, 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 cdc mutants still continue to grow, they become abnormally large. By contrast, non-cdc mutants, 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 reserves run out.

In Animal Cells
The Decision point at late G1 is called: Extracellular Growth Restriction Point = START Factors signaling. If growth factor is not available Go stage cells enters Go, the quiescent stage of cell cycle.

metabolically active cease growth & proliferation Less protein synthesis.

Wound Blood Platelets

Skin fibroblasts activated to grow if there is wound

PDGF

Clotting

Signals proliferation of fibroblasts/skin cells near injury

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

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.

In Human
G1 S G2 M
Schizosaccharomyces pombe (Fission yeast) Yeast system Saccharomyces cerevisiae (Budding yeast)

Mutation

in cell division cycle genes (cdc genes)

Cell Cycle
= Life cycle of a cell=
Birth Growth Reproduction Divided into different Phases G1 S G2
9-12 hr G1 = growth & preparation or DNA duplication 10-12 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 bone marrow stem cells differentiate to blood cells 16 steps

Death M Go

Synthesize RNAs & Proteins

In adult person

Driving forces are Specific Proteins present in the cell Controlled by Cell Division Cycle genes = cdc genes

(Kinases & Cyclins)

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

Cell cycle:

An orderly sequence of macromolecular events that allow duplication of cellular contents and cell division leading to production of two daughter cells each containing identical copy of genetic materials of the parental cell.

Cell division and cell cycle occur in proper order and with high fidelity. Regulation of cell cycle is critical for normal development and loss of control leads to CANCER. Cell replication is primarily controlled by regulating timing of nuclear DNA replication and mitosis. The master molecules that control cell cycle events are a number of Heterodimeric Protein Kinases.

Regulatory submit Catalytic submit

Cyclins Cyclin dependent Kinases (cdks) (regulate through phosphorylation)

DNA 2 no levels

Chromosomes
proteins

Protein (nuclesome) Histones & Chromosomal

RNA

Interphase = (G1 s G2) Mitosis (M) Prophase Metaphase

Anaphase

Telophase

 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 Quiescent Stage. .. Anaphase Promoting Complex Cyclosome PRO Meta Anaphase Telaphase remain longtime without dividing

Cell cycle control Check Points = Control system blocks

progression through these check points if it detects problems inside or out side the cells. 1. Restriction point/Start (Commits cell cycle entry) G2/M check point Late G1 G1/s

2.

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) & cytokines) leads to completion of mitosis

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

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

1.

G1/s cyclins activates cdks at G1 Start commit entry to cell cycle. (G1/S-cdk) complex. S Cyclins bind cdks soon after progression through Start. (S cdk) complex. M Cyclins activates cdks that stimulates entry into Mitosis at G2/M check points (M cdk) complex. G1 cyclins Govern the activities of the G1/S cyclins & controls progression through start in G1.

2.

3.

4.

In vertebrates: 4 Cdks G1 cdk, G1/S cdk, S cdk and M - cdk

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.

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. Binding of cyclin causes T loop to move out of active site causing partial activation of Cdk 2. Phosphorylation of Cdk 2 by Cdk activating kinase (CAK) at threorine residue in T loop activate the enzyme fully and allow the enzyme to bind its protein substrates.

B.

C.

Cdk activity can be suppressed by inhibitors

Phosphorylation of Cyclin Cdk complex by a protein kinase Wee inhibits Cdk activity. Dephophorylation of Cdk sites by phosphatase known as cdc Cdk activity.

25 increses

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 final stages of Cell Division.

APC/C (Anaphase promoting complex/cyclosome ubiquitin by gase enzyme) Ubiquitylation & destruction of

Securin

protects proteins linkages that hold sister chromatids together.

Separates sister chromatides (unleashes anaphase) Destroy S and M cyclins. Dephosphrylation of proteins & Cdk targets. Cytokinesis Cell Division -

APC/C remains active in G1 Late G1

Cdk remains inactive

G/S Cdk gets activated

APC/C turn off.

Allow Cyclin accumulation

Start Next cell cycle

Cyclin cdk complex 1. 2. 3. 4. G1 cdk G1/S cdk S cdk M cdk cdk4 + cdk6 + Cyclin D cdk2 + Cyclin E cdk2 + cdk1 + Cyclin A cdk1 + Cyclin B

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 other organcells and macromolecules & incerese cell mass. Segrate the all copies precisely into two genetically identical cells.

How to study Cell Cycle?

Human Fibroblast 25 40 divisions Replicative cell senescence

Cells in culture Transfetion Immortalized cell lines mutation used for cell cycle study

genetically homogeneous cells.

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

Direct visualization of growing cells at different stages 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.

 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.

II

III

II

How to study Cell Cycle?......... By measuring DNA content 2n/2c value by 4C in G2 phase by Flowcytometer (FACS) G1 (Fluorescence activated cell sorting)

In each cell First 1. Second 2.

Amount of fluorescence = amount of DNA Present unreplicated complement of DNA Fully replicated complement of DNA (Twice G1 DNA content) Intermediate amount of DNA G1 G2/M phase

Third 3.

S - Phase

Analysis of DNA content with a flow cytometer

This graph shows typical results obtained for a proliferating cell population when the DNA content 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, and those 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.

Dissection of Cell cycle using yeast mutations

Two yeast species used in cell cycle study (1% of gene) 1. Fission Yeast = Schizosackharonyces pombe (used in producing beer) 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. Search mutation in yeast that inactivate genes encoding essential cell cycle regulatory genes = cdc gene

Temperature sensitive Mutation Low Temperature Normal Prolif (No Mutation cdc) Yeast flow in different phases of cell cycle. High Temperature Mutation (cdc Mutation) by seeing the buds/size of the bud

Example:

cdc15 mutant at high temperature complete upto anaphase but can not complete anaphase sepration and cytokinesis. The buds became large & remain late M phase. day cycle Monthly cycle Yearly cycle (circa.)

Cell cycle = Circulation Rhythm day night Chronobiology Chronopharmacology

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

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

Cell Cycle Regulation

Protein Phosphorylation & Degradation of proteins control passage through cell cycle.
1. Concentration of Cyclins the regulatory submits of heterodimeric protein kinases (Cdks) catlytic submit that control cell cycle events increase or decrease as cells progress through cell cycle. CDKs have no kinase activity unless they are associated with cyclins. Associated cyclin determines which proteins to be phosphorylated by the cyclin-cdk complex.

2. 3.

Current model for regulation of the eukaryotic cell cycle Passage 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 pathway polyubiquitinate 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 of proteindegradation. Proteolysis of anaphase inhibitors inactivates the protein complexes that connect sister chromatids at metaphase (not shown), thereby initiating anaphase.

Cell Cycle Regulation

3 major cyclin cdk complexes controls:
1. G1 cyclin cdk complex express first to stimulate replication at late G1

2. 3.

S cyclin cdk complex Mitotic cyclin cdk complex

Activate Transcription factors

Promote Transcription of Genes codes for 1. Enzymes for DNA synthesis 2. Sphare cyclins 3. S phase cdks S phase cyclin cdk complex is checked by inhibitors.

At late G1 G1 cyclin cdk complex induce degradation of inhibitors by phosphorylation & ubiquitination by SCF ubiquirtin lygase.

Activates/initates Replication of DNA

Phosphorylate regulatory sites of proteins DNA prereplication Complex

Releases active S phase Cyclin cdk complexes

Prevents assembly Of pre replication complex

Assembled at Replication Origin

Because of this inhibition Each chromosome is replicated once So proper chromosome number is Maintained in daughter cells.

 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.

Once activated by dephosphorylation at inhibitory sites, mitotic cyclin CDK complexes phosphorylates multiple proteins that lead to
Chromosome condensation Retraction of nuclear envelope Assembly of mitotic spindle apparatus Alignment of chromosomes at metaphase plate

1. 2. 3. 4.

 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 all chromosomes and attached spindles microtubules and aligned at metaphase plate. Following Securin degradation initiation of Anaphase occurs by freeing sister chromatids to seggregate to opposite poles.

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. 2. Inactivation of protein kinase activity of mitotic CDKs Active protein phosphasases remove phosphates from specific proteins. Separated chromosomes start decondensing. Nuclear envelope reforms around daughter nuclei Golgi apparatus reassembles at Telophase Cytoplasm devides at Cytokinesis resulting in two daughter cells.

3. 4. 5. 6.

 During the early G1 of next cell cycle, phosphatases dephosphorylates proteins that Pre-Replication complex. Because of dephosphorylation at G1, a new prereplication 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 Sphase and mitotic cyclins during ensuing cycle.

Passage through three critical cell cycle transitions 1. 2. 3. G1 S phase Metaphase to Anaphase 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.

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.

(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.

(B) Control of proteolysis by SCF

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.

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.

Summary of the Major Cell-cycle Regulatory Proteins

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 (Self renewal capacity) Differentiated to

No function

Skin cells

Nerve cells

Blood cells

Muscle cells

Immune cells

 Development of organs & tissues in multicellular organisms depends on specific pattern of mitotic cell division. A series of such cell division in a family tree called cell lineage which can trace the birth order, developmental potential and differentiation to specialized cell types. Cell lineages are controlled by both internal and external factors. cells cell signals and environmental factors. A cell lineage begins with Stem Cells the name derives from plant stem. It ultimately form terminally differentiating cells (Nurons, skin, liver) which is a irreversible (??) process.

 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.

 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.

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.

8 cell stage mice

cell 8 cells are totipotent.

Neurons (transmitting) Propagating electical signals Provides electrical insulation

Particular Progenitor Cells divides Gial cells Stem Cells Neurons Neurotransmitter Dopamine Transplant embroyonic neurons in Parkinson patient.

Dopamine producing neurons No rejection by immune system

Differentiation: Stem cells

so fertilization of the egg takes place in the oviduct the fertilizes zygote travels to the uterus for implantation along the way the zygote begins to divide (mitosis) 2-cell, 4-cell, 8-cell embryonic stages etc. the embryo reaches a stage called the morula = ball of small cells (embryo has not enlargened) by the end of the first week the second embryonic stage the blastocyst - forms

the blastocyst is a hollow ball of cells containing an outer rings of progenitor cells = trophoblast and an inner mass of cells at one end of the embryo = inner cell mass it is these ICM cells that are the source for the derivation of embryonic stem (ES) cells

Differentiation: Embryonic Stem cells

the ES cells are said to be totipotent have the ability to specialize or differentiate into ALL cells of the embryo the blastocyst then begins a process of differentiation and these ES cells form populations of stem cells with more restricted potentials the ES cells first differentiate into two layers called the embryonic disc divides the blastocyst cavity into an amniotic cavity and a yolk sac (primitive hematopoietic organ) these two layers then continue to differentiate into the three germ layers of the embyro
ectoderm, mesoderm and endoderm

the formation of these germ layers marks the gastrula embryonic stage

Germ Layers
the ectoderm, mesoderm and endoderm are thought to be made up of stem cells with a more restricted phenotype when compared to ES cells BUT still capable of 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 around these tissues results in specific developmental events = patterning patterning requires the exposure of cells to a succession of signals and subsequent activation of their associated pathways

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 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 of Phase Variation.

 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 by occasional 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 Promoter located within the invested segment.

 If The promoter in one orientation, the bacteria can synthesize one type of Flagellin while Promoter in other orientation, they synthesize the other type of Flagellin protein. Inversions occurs only rarely and that is why bacteria generally have one or the other type of Flagellin. This type of inversion by phase variation protects bacterial population against the immune response of the host. If the host makes antibodies against one type Flagellin, some bacteria which have synthesized other flagellin by gene inversion will be able to survive and multiply.

 Wild type of bacteria is exhibit phase variation while laboratory 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 gene conversion instead of gene inversion. Transfer DNA sequences from a library of silent gene cassettes 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. Cell Memory: When cells are commited to differentiate to a specific cell type, cells remembers the changes in gene expression for several generations.

Other changes in gene expression in eukaryotes & bacteria are transient. +Tryptophan Example: Tryptophan gene in Bacteria (Sal monella) switched off Remove Trisptophan Gene is switched on. Bacterial cells will not have any memory that cells were exposed to Tryptophan

DNA reaggangements mediate phase Variation. Cell differentiation occur in higher eukaryotes without detectable change in DNA sequence.

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 Recombination enzyme Leads to change in expression of cell surface protein = Flagellin 2 genes are responsible.

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 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.

Switching Gene expression by DNA Invension in Bacteria

Alternating transcription 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 one orientation, 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 H2 or 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.

Switching gene expression by DNA inversion in bacteria.

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.

Saccharomyces cerevisiae, a single cell eukaryote which can exist in 3 distinct forms:
1. 2. 3. Haploid a type Haploid type 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 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 Mat 1 and Mat 2. Mat a1 protein has no effect in the a type haploid cells which has produced this but important in Diploid cell. In contrast, Mat 2 protein acts in cell as Transcriptional Repressor that turns of a specific genes.

 Mat 1 protein acts as a Transcriptional Activator that turns on the specific genes. Once cells of two mating types have fused, the combination of Mat a1 and Mat 2 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 cell type - specific transcription factors 1, 2, a1) encoded by MAT Locus and act in combination with general transcription factor MCM-1mediate cell type specific gene expression. asg = a specific gene/mRNA sg = specific gene/mRNA hsg = Haploid specific gene/mRNA

2.

Thus the actions of these three transcription factors can set the yeast cell on specific differentiation pathway cell minuting in a particular cell type. Microarray study established expression and repression of many genes that control cell characteristics. Activity of MCM-1 is determined by its association with 1 and 2 transcription factors. As a combinatorial action, MCM 1 promotes transcription of genes and repress a specific of genes in cells. specific

3.

4.

5.

Three gene regulatory proteins (Max 1, Max 2 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 cells express a set of haploid-specific genes (hSG) and either a set of -specific genes ( SG) or a set of aspecific 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 Max 1 protein is a gene activator protein, whereas the Max 2 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, Max 2 and Mata 1 from a heterodimer (shown in detail in Figure) that turns off a set of genes (including the gene encoding the Max 1 activator protein) different from that turned off by the Max 2 and Mcm1 proteins. This relatively simple system of gene regulatory proteins is an example of combinatorial control of gene expression.

Control of cell type in yeasts.

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 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 off by Mcm 1 & Mat 2 proteins. This is combinational control of gene expression.

MCM 1 First member of MADS family of transcription factor (axrormym of 4 factors of the family) (Mini Chromosome Maintenance gene 1) 1. 2. 3. 4. MCM 1 from budding yeast. Agamous from Arabidopsis thaliana. Deficiens from Snagdragon SRF from Human

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 -specific genes and a-specific genes, respectively. a.) In a cells, MCM1 stimulates transcription of a-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 of a-specific genes. The 2-MCM1 complex also is produced in diploid cells, where it has the same blocking effect on transcription of a-specific genes.

Circadian clocks are based on Feedback loops in gene.

Central role as time keep on of mammals SCN Suprachiasmatic nucleus cells in hypothalamus.

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 diurnal pattern of 24 hr. cycles

Gene Regulatory Proteins determine cell type in yeast Single cell Eukaryote. Bakers yeast = Saccharomyces cerevisiae 1. Haploid 2. Haploid a 3. Diploid (a/ )unable to mate but sporulate. Mat locus Mating type Mating factor a type type &a

Mat locus Mat locus

Mat a1 Mat 1 Mat 2

Gene regulatory protein 1. Haploid a Mata (No effect)

Cell type/Yeast

Set of genes controlled by Mat

2. (Haploid) Mat 1 Mat 2

3. Mat 2

/a Mat a1

/a (Diploid)