molbiol 2011-07-chromosomes-cell-cycle

Chromosomes and Mitosis

Lecture 6

1 Chromosomal Basis of Heredity• A gene is a unit

of heredity• Genes are

carried on DNA• DNA is

contained within chromosomes as chromatin

Chromosomes replicate during cell division

The chromosome complement

Chromosome analysis

Cri Du Chat results from loss of a small piece of chromosome 5

Gene Map

Chromosome pairs

Non-identical genes

Sex chromosomes

• These determine the sex of an individual– Two X chromosomes make a female

– One X and one Y a male

Two types of Cell Division

• Cells divide for two reasons– To create genetically identical

copies of themselves• This is

mitosis

– To create gametes that contain half of the chromosomes of the original cell

• This is meiosis

46

46 46

46

23 23 23 23

The Cell Cycle

S phase

ReplicationCondensation

Schematic

DNA replication

Duplex DNA begins Replicating

Replication bubbles merge creating two duplexes

Mitosis

The stages of Mitosis

Prophase Detail

Prometaphase

Metaphase

Anaphase

Telophase

The sum total of the process

Karyotypes

Chromosome Length

Chromosome appearance

Meiosis and Gametogenesis

Somatic and Germline cells

• Development of a fertilized egg into an adult results in two distinct types of cells– Somatic cells

• These create all tissues and organs of the adult except for cells destined to become sperm or egg

• They can only undergo mitosis

– Germline cells• The final differentiated form of these cells are mature gametes:

the sperm and egg

• These cells undergo mitosis until gametogenesis– They then undergo meiosis

Meiosis

Meiosis is required for gametogenesis

Meiosis ISomatic cells

Germline Cells

Interphase I and Prophase ILeptotene

Prophase IZygotene

Prophase IPachytene

Prophase IDiplotene

Recombination

And on the molecular level

Metaphase I and

anaphase I

Meiosis I is the reduction division

Non-disjunction

Telophase I

Cytokinesis

sperm formation oocyte formation

Meiosis II

A comparison of meiosis and

mitosis

Mitosis Meiosis

Chromosome number

Maintains Reduces

Nuclear Divisions 1 2

Cells resulting 2 4

Cells involved Somatic Germline

Relationship to Gametogenesis

Sperm and Egg formation

Gametogenesis

Fertilization

• Entry of a single sperm into an egg prevents entry of other sperm

• The DNA of sperm and egg are initially kept separate in “pronuclei” of the zygote

• Timing of a pregnancy extends from the “last menstrual period” (LMP) rather than the time of fertilization

Mitotic Non-disjunction

Cell cycle and apoptosis• Cells undergo 3 controlled processes

– The first two are part of the cell cycle, the last an exit from the cell cycle

– Division (the cell cycle)– Quiescence

• This is where most of the work of being a cell lies– During division the energy of the cell is devoted to making a new cell

– Death• This can be a normal process creating a final functional form or an induced

suicide– Epithelium and reticuloendothelial cells undergo active transitions towards

terminally differentiated states in which the final forms are unable to divide» The stratum corneum consists of cells that have become bags of

crosslinked keratin protein with no internal metabolism– Suicide can be induced because the organism senses a threat to the entire

organism» Infection, cancer, avoidance of autoimmunity

Control of entry into cell cycle and apoptosis

• Cell cycle is initiated by phosphorylation of transcription factors

• These activate transcription of a set of proteins known as cyclins

• The appearance of cyclins is progressive and determines the types of proteins that will be phosphorylated at a particular point during the cell cycle

Cyclins and CDK’s• CDK levels don’t

change while cyclins are destroyed at the end of each phase

• There are 3 general groups of each– G1 cyclins

• Cyclin D

– S-phase cyclins• Cyclin A

– G2 cyclins• Cyclin B (maturation

promoting factor MPF)

– Cyclin E is shared between G1 and M phase

– Cyclin A is shared between M phase and G2

Cyclins bind CDK’s • CDK’s are Cyclin

Dependent Kinases• Association with cyclins

activates their kinase function– A cyclin tethers a target

protein to the CDK

• The targets of CDK’s are transcription factors among other proteins– CDK’s are serine/threonine

kinases

The exit from Go

• Go is a quiescent state• Activation of G1 CDK

occurs due to a rising level of G1 cyclins

• G1 cyclins are transcriptionally activated by growth factors

Events during G1

• A rising level of G1 cyclins increases the activity of G1 CDK’s

• CDK’s in turn activate proteins and in turn genes that prepare the cell to begin DNA replication

• At the G1 S boundary, the cell encounters a checkpoint

G1/S checkpoint

• This is controlled by the activity of the transcription factor E2F– E2F is a family of related proteins (E2F 1 to

E2F5)

• E2F is found complexed throughout the cell cycle to another family of proteins: Rb– At the G1/S checkpoint, Rb is

phosphorylated by CDK2/cyclinA– E2F is freed from sequestration and activates

transcription at genes containing an E2F consensus sequence

And those genes are• Three groups

– Cell cycle regulators• Cyclin A• E2F, Rb, myc, myb

– Note that these are not all positive regulators of cell cycle

– Enzymatic machinery for DNA synthesis

• DNA polymerase• PCNA• Enzymes involved in

nucleotide metabolism

– DNA synthesis regulators• Enzymes that recognize the

origins of replication for example

Other Checkpoints

• These monitor the completion of DNA synthesis– The presence of Okazaki

fragments prevents entry into G2

• DNA damage – This occurs before, during and

after completion of S phase

• Spindle attachment– Failure to attach spindle to

centromere results in blockage of mitosis at metaphase

– Failure to align the spindle during cytokinesis results in blockage at anaphase

Downregulation of cyclin influenced

CDK activity

• This is accomplished through proteolysis of the cyclins– G1 phase cyclins disappear during S

and G2 phase

– M-phase promoting factor (CDK2 + cyclin B) concentrations rise just prior to onset of mitosis

• Cyclins associated with MPF are degraded by anaphase promoting complex

– Cyclin B levels peak at G1/M

» Degradation during anaphase

– APC promotes polyubiquitination of cyclin B

– Ubiquitinated cyclin B is degraded by a proteosome

• Cyclin transcription is also turned off and the mRNA is unstable– So no new cyclin is made until

transcription is restored

cyclin B

cyclin A

ribonucleotidereductase

Mitosis MitosisInterphase Interphase

Time

Newly synthesized proteins labeled with 35S-methionine:

MPF activates APC which

ubiquitinates cyclin B

In the overall• Stimulated entry into G1

results in appearance of an initial level of cyclins that promote the progressive activation of genes enabling the cell to synthesize DNA

• A series of progressive steps result in– Activation of genes further

into the cycle

– Degradation of cyclins that promoted earlier steps

– Passage through checkpoints that insure mechanistic fidelity of each step

Apoptosis (apo – toe – sis)

• This is programmed cell death– Distinguish it from necrosis

– Necrosis results from traumatic forces outside the cell

– Necrotic tissue provokes inflammation as the immune system moves in to clear out damaged and dead cells

• Apoptosis is an ordered stepwise self-destruction that permits surrounding cells to utilize the breakdown products of the dead cell– There is no inflammation involved

The apoptotic cell

• Mitochondria break open

• DNA fragments in a regular way

• The cell loses a regular shape– Undergoes blebbing– This is an irregular

bubbling appearance of the plasma membrane

The mechanisms of apotosis

• Can be classified as externally or internally signaled

• One internal route involves p53

• p53 is a transcription factor that is involved in cell cycle control and sensing the presence of DNA damage

• The central role p53 plays is at the G1/S checkpoint

P53 controls entry into S-phase• P53 can sense DNA damage by binding mismatches

• In the presence of damage, p53 activates transcription of p21– P21 binds and inactivates CDK2-cyclin E complexes

– The complex is unable to phosphorylate Rb and free E2F

– Thus entry into S phase is inhibited

– If the damage is repaired, p53 levels and p21 levels drop and S phase ensues

But if the DNA damage is extensive

• P53 induces apotosis by activating transcription of Bax– BAX protein competes with BCL-2 to form

pores in mitochondrial membranes• BCL-2 prevents the release of cytochrome c from

mitochondria into the cytoplasm• BAX permits release of cytochrome c

– When released, cytochrome c stimulates caspase activation

The caspases

• These are proteolytic enzymes that are held in check by external or internal inhibitors

• Activation results in an explosive proteolytic cascade– Caspase 9 cleaves and

activates other caspases

– The caspases also activate quiescent nucleases

External apoptotic mechanisms

• Involve external “death signals”

• Cells may be recognized as a threat to the whole organism– The immune system moves

in to kill them

– One mechanism of killing involves a command to the cell to initiate apoptosis

Fas/Fas ligand signaling

• Fas ligand (FasL) is a membrane bound cell surface protein

• It binds to Fas receptor• Binding results in trimerization

and activation of APAF• APAF in turn activates caspase 8

by proteolysis of a caspase 8 zymogen– Caspase 8 cleaves a BCL-2 family

member BID– BID translocates to the

mitochondria and binds BAX– Bax permits leakage of cytochrome

c and activation of the caspase 9 cascade via APAF-1 again