Cell Cycle, Mitosis, Meiosis, and Recombination

Question 1

Outline the basic phases of the cell cycle and the processes that take place during each phase.

Answer 1

Cell division is the process by which a parent cell divides into two or more daughter cells by progression through the cell cycle.

G0 - A resting phase where the cell has left the cycle and has stopped dividing (quiescent / senescent).

G1 - Growth phase during which proteins and RNA are synthesised. Each chromosome exists as a single double stranded helix - at no point is DNA synthesised in this phase. At the G1 checkpoint - the restriction point - the cell is committed to division and moves into the S phase. 9-12h.

S - DNA synthesis replicates the genetic material. Each chromosome now consists of two sister chromatids. 6-8h.

G2 - Cell continues to grow. The G2 checkpoint ensures enough cytoplasmic materials necessary for mitosis and cytokinesis. 2-5h.

Note: G1, S, and G2 collectively form interphase.

M - The cell stops growing. Nuclear division (mitosis) followed by a cell division (cytokinesis). The Metaphase checkpoint in the middle of mitosis ensures that the cell is ready to complete cell division. 1h.

*Average duration for lymphocytes in culture is ~24 hours; this varies for different cell types.

Question 2

What is the average duration of the cell cycle for lymphocytes in culture?

Answer 2

The average duration for lymphocytes in culture is ~24 hours; this varies for different cell types.

Question 3

What is mitosis?

Answer 3

Mitosis is part of the cell cycle in which chromosomes in the nucleus are separated into two identical sets of chromosomes contained within their own nucleus.

Question 4

What are cell cycle checkpoints?

Answer 4

Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions ensuring critical events are completed with high fidelity.

Question 5

How is the cell cycle regulated?

Answer 5

The cell cycle is regulated by heterodimeric protein kinases composed of:
Cyclins
Cyclin-dependent kinases (CDKs)

Question 6

What are cyclins?

Answer 6

Cyclins - Form the regulatory subunit and have no catalytic activity

Question 7

What are Cyclin-dependent kinases (CDKs)?

Answer 7

Inactive in the absence of a partner cyclin. Becomes the catalytic subunit (serine/threonine protein kinases) of an activated heterodimer which phosphorylates target proteins to orchestrate coordinated entry into the next phase of the cell cycle

Question 8

When are CDKs and cyclins expressed?

Answer 8

CDKs are constitutively expressed whereas cyclins are synthesised at specific stages in response to various external stimuli / molecular signals.

Question 9

Why are checkpoints important?

Answer 9

Checkpoints are essential to ensure that the cell cycle halts if chromosomal DNA is damaged or critical processes such as DNA replication or alignment have not been completed properly.

Question 10

What are the three main checkpoints?

Answer 10

G2/M Checkpoint

G1/S (restriction) Checkpoint

Metaphase / Spindle Checkpoint

Question 11

G2/M Checkpoint

Answer 11

CDK1 is activated by phosphorylation and de-phosphorylation of specific amino-acid residues by Cyclin-Activating Kinase (CAK, as well as the inhibition of the wee1 protein (which inhibits CDK1)

Enables CDK1-cyclin B formation (aka MPF)

Allows G2-M phase transition

Question 12

G1/S (restriction) Checkpoint

Answer 12

Cell growth enables formation of the CDK4/6-cyclin D

Phosphotylates retinoblastoma protein

Relieves inhibition of E2F transcription factor

Cyclin E now expressed, binds to CDK2

Question 13

Metaphase / Spindle Checkpoint

Answer 13

Chromosomes assemble on metaphase plate

Anaphase-promoting complex (APC) activated

Degrades cyclin B = MPF disassembly

Relieves inhibition of ‘separase’ which cuts cohesion

Sister chromatid separation = anaphase entry

Question 14

How do Cytogeneticists manipulate the cell cycle?

Answer 14

Mitogens - used to induce division of resting cells:(PHA, pokeweed, concanavilin A)

Synchronisation - Inhibitors block cell cycle during S phase by slowing/stopping DNA synthesis (FudR/uridine, Thymidine).

Block released after 16-22h - cells continue through G2 together

Mitotic arrestants - stop division during mitosis (colchicine/Colcemid®). Prevents spindle fibre apparatus formation. Stops cell at metaphase.

Question 15

What does p53 do to checkpoints?

Answer 15

p53 (“the guardian of the genome”) plays an important role in controlling progression through G1/S and G2/M checkpoints. P53 is a critical component of DNA damage checkpoints

DNA damage -> Activated p53-> Inhibits progression through checkpoint

Question 16

What is meiosis?

Answer 16

Meiosis is a specialised type of cell division that reduces the chromosome number by half.

Question 17

What are the broad stages of meiosis?

Answer 17

Interphase (G1, S and G2 )- this is followed by two rounds of cell division (meiosis I and meiosis II) to produce four potential daughter cells, each with half the number of chromosomes as the original parent cell. The daughter cells are not genetically identical to the parent cells (unlike mitosis).

Question 18

Describe Prophase I of meiosis

Answer 18

Prophase I
Leptotene
Nuclear chromatin begins to condense/become visible. Chromosomes are unpaired fine threads consisting of 2 tightly bound sister chromatids (AKA “A string with beads.”)

Zygotene
Maternal and paternal homologues pair to form bivalents- held together by formation of synaptonemal complex. In males, X and Y condense to form sex vesicle. The 2 sets of sister chromatids is termed a bivalent and the connecting points are chiasmata.

Pachytene (early)
All homologues have paired (synapsis). Bivalent at this stage is called a tetrad.

Pachytene (late)
Chromosomes thicken, cross over and recombination of genetic material occurs. Two non-sister chromatids cross over, the other 2 remain unaltered. Approx 60 (sperm) to 90 (ova) crossover events occur per cell with at least one per chromosome arm.

Diplotene
Homologues start to separate (desynapsis) but are held together by chiasmata- may resemble cross-like appearance. Sex vesicle disappears and X and Y appear associated end to end. The small chiasma formed is enough to keep X/Y chromosomes paired on spindle during metaphase- resulting sperm cells will have either X or Y chromosome.

Diakinesis
Bivalents are more contracted. Nuclear envelope breaks down. Oocytes reach this stage at ovulation

Question 19

Describe Metaphase I of meiosis.

Answer 19

Spindle formed, bivalents align along metaphase plate. Organisation critical to ensure one copy of each chromosome is received in each new nucleus. In females, spindle is off centre and one resultant cell will contain more cytoplasm. X and Y may separate to form univalent

Question 20

Describe Anaphase I of meiosis.

Answer 20

Homologous chromosomes drawn apart. Chromatids remain together

Question 21

Describe Telophase I of meiosis.

Answer 21

Chromosomes at poles. Haploid daughter cells formed. In females, the cell with larger amount of cytoplasm is called secondary oocyte and the smaller one is first polar body.

Question 22

Describe Prophase/ Metaphase II of meiosis.

Answer 22

Cells pass directly from meiosis I to metaphase II with no real prophase II. Occurs immediately in males. In females it is co-ordinated with ovulation and fertilisation. Nuclear envelope breaks down, new spindle formed, chromosomes (consisting of 2 chromatids) align.

Question 23

Describe Anaphase II of meiosis.

Answer 23

Separation of centromeres, migration of sister chromatids to opposite poles

Question 24

Describe Telophase II of meiosis.

Answer 24

Further cell division occurs forming 2 haploid cells (4 in total). In secondary oocyte, spindle is again off centre resulting in a large cell (ovum) and second polar body. Two more polar bodies may arise from the first but the ovum is the only viable gamete.

Question 25

What is Gametogenesis ?

Answer 25

Gametogenesis occurs by meiotic division of diploid gametocytes into various gametes

Begins in utero for females (Oogenesis) and puberty in males (Spermatogenesis)

Question 26

What is MI (meiosis 1) ?

Answer 26

MI (meiosis 1) – random independent assortment of chromosome pairs and crossover enables genetic recombination prior to separation

Question 27

What is MII (meiosis 2)?

Answer 27

MII (meiosis 2) – separation of chromatids (to haploid state)

Question 28

How diverse can human gametes be?

Answer 28

Independent assortment of maternal and paternal homologues in human cells creates the possibility of 2^23 (8.4 million) genetically different gametes

Recombination of genetic material via crossing over forms daughter cells that contain composites of both parental genes; therefore, the actual number of genetically different gametes is infinite.

Both of the above increase genetic diversity between daughter cells

Question 29

What does genetic recombination involve?

Answer 29

Recombination takes place during prophase I stage of meiosis. Homologous chromosomes line up in pairs and swap segments of DNA, i.e., “crossing over”.

Genetic recombination involves :
Alignment of two homologous DNA strands
Precise breakage of each strand
Equal exchange of DNA segments between the two strands
Sealing of the resultant recombined DNA molecules through the action of enzymes called ligases.

Question 30

What are chaisma?

Answer 30

Recombined homologs are physically connected at specific points which mark the location of the crossover. These points are known as chaisma. There are on average 55 chiasmata per cell in male meiosis and possibly 50% more in female meiosis. Chiasmata are also thought to be essential for correct chromosome segregation in meiosis I.

Question 31

Give an overview of meiotic recombination

Answer 31

Recombination is initiated by DNA double-strand breaks

Ends are rapidly processed to form long single-stranded tails with 3’-termini

Inter-homolog single-end invasions (SEI) by the single-stranded tails takes place

SEI results in double-Holliday junctions (DHJ) formation

DHJs are resolved into cross-over products

Question 32

Potential consequences of recombination

Answer 32

Non–allelic homologous recombination (NAHR)

Single gene disorders (Mendelian disorder) - where recombination results in the deletion or duplication of a single gene (autosomal dominant/recessive and X-linked) such as Rubinstein–Taybi

Contiguous gene disorders – caused by chromosome abnormalities where several genes are deleted/duplicated resulting in alteration to normal gene dosage such as Wilms’ tumor Aniridia Genitourinary abnormalities and mental Retardation (WAGR)(11p13)

Segmental aneuploidy (aneusomy) syndromes – refers to the relative excess or deficiency of specific chromosome regions; contiguous gene syndromes with well-defined phenotypes such as Di George/VeloCardioFacial Syndrome (VCFS)/22q11.2 deletion or Prader-Willi Syndrome (PWS) (paternal 70%/maternal 25%/genomic imprinting defects), these recur due to non-allele homologous recombination between low copy repeats