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

The Cell Cycle

S phase Replication Condensation 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 I Somatic cells Germline Cells

Interphase I and Prophase I Leptotene

Prophase I Zygotene

Prophase I Pachytene

Prophase I Diplotene

Recombination

And on the molecular level

Metaphase I and anaphase I

Meiosis I is the reduction division

Non-disjunction

Telophase I

Cytokinesis sperm formationoocyte formation

Meiosis II

A comparison of meiosis and mitosis MitosisMeiosis Chromosome number MaintainsReduces Nuclear Divisions12 Cells resulting24 Cells involvedSomaticGermline

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

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