L1 - introduction to cell cycle

Question 1

What is the cell division cycle made up of?

Answer 1

G1
S
G2
M

Has a driven order

Question 2

What are GAP phases?

Answer 2

Occur in-between DNA synthesis and mitosis

G1
G2

Question 3

What is the difference between S. cerevisiae and S. pombe?

Answer 3

Are very different from each other

S. cerevisiae grows by budding
S. pombe grows by elongation

Are both easy to manipulate

Are evolutionary very far apart - ~600 million years

If we can show similarities between the 2, probably means humans have the same similarity

Question 4

What are permissive conditions?

Answer 4

Conditions under which the mutant strain will grow

Question 5

What are restrictive or non-permissive conditions?

Answer 5

Conditions under which the mutant strain will not grow

Question 6

What is a temperature sensitive mutation?

Answer 6

A mutation that shows a phenotype at a temperature at which the wild type grows

Question 7

What are the types of temperature sensitive mutations?

Answer 7

ts - temperature sensitive
the yeast mutant doesn’t grow at high temperatures (35-37) but will grow at normal permissive temperatures (28-30)

cs - cold sensitive
the yeast mutant doesn’t grow at low temperatures (15-20) but will grow at the normal permissive temperatures (28-30)

Question 8

Why is temperature important in conditions?

Answer 8

Most likely affects the stability/folding of the mutant protein influencing the function of the protein

Question 9

Life cycle of S. cerevisiae

Answer 9

Nucleus migrates to a new bud

Formation of the mitotic spindle across the bud neck

Mother cell is bigger than daughter cell when it divides
• creates problem for the cell
• can see exactly where the cell is in the cell cycle

Question 10

S. CEREVISIAE
Morphology of asynchronous cultures of a wild type strain, a cdc28-ts mutant strain and a noncdc-ts mutant strain growing at the permissive temperature (28 degrees)

Answer 10

Wild type strain - cell number keeps increasing

Cdc28-ts mutant strain - cell size almost doubles before stopping

Noncdc-ts mutant strain - no chance from the wildtype

If the mutation is involved in the cell cycle, all the cells will get stuck in the same place - stop passing a specific place in the cycle

Question 11

Life cycle of S. pombe

Answer 11

Instead of forming a bud, it elongates - diameter doesn’t change but length does

Nucleus tends to lie in the middle of the cell

Cell divides equally in mitosis

Question 12

S. POMBE
Morphology of asynchronous cultures of a wild type strain, a cdc28-ts mutant strain and a noncdc-ts mutant strain growing at the permissive temperature (28 degrees)

Answer 12

Wild type strain - cell number keeps increasing

Cdc28-ts mutant strain - cell size almost doubles before stopping

Noncdc-ts mutant strain - same as the wild type

Question 13

What are Cdc mutations?

Answer 13

Cell division cycle mutations

Question 14

What do Cdc mutations show?

Answer 14

• Define genes necessary for transit through specific points in the cycle
• The block marks the point beyond which the cells cannot progress
• Block point may not be the same point where the wild type gene normally acts in the cycle
• Mutations isolated are conditional mutations
• Different allele identification may identify several functions of a protein

Question 15

What is a conditional mutation?

Answer 15

A mutation that has wild-type (or less severe) phenotype under certain “permissive” environmental conditions and a mutant phenotype under certain “restrictive” conditions

phenotype may not be the same as that of the null
• partial functions may leak through or change the function rather than destroy it

Question 16

Problems with Cdc mutations

Answer 16

Gene pathway redundancy

Mutation is very rare

The Cdc gene has many cell cycle functions

Question 17

FACS analysis

Answer 17

Fluorescent Activated Cell Sorter

1) Cells are stained with a fluorescent dye which binds DNA
2) Cells are then analysed in the FACS to study the status of the DNA content of the cell population in the cell cycle
3) Fluorescent signal detected by the FACS is directly proportional to the DNA content of the cell

Question 18

Why is synchrony important in studies of the cell division cycle?

Answer 18

Determining cell cycle fluctuations in protein and RNA synthesis

Examining potential cell cycle protein modifications

Examining protein associations

Examining localisation of proteins

Question 19

How are cell cultures synchronised?

Answer 19

1. Centrifugal elutriation which separates cells bases on cells
   • Cells tend to be smaller at the beginning of the cycle so can be centrifuged out and then synchronised
2. Shifting Cdc-ts mutants to the restrictive temperature then releasing them from the block
3. Chemical treatments - eg. hydroxyurea
4. In S. cerevisiae, alpha mating factor blocks ‘a’ type haploid cells in G1 phase

Question 20

Problems with synchronisation

Answer 20

Physiological consequences of perturbing (causing anxiety) the cell cycle

Question 21

What are the 2 models for the cell cycle regulation?

Answer 21

Sequential steps

Sequential steps and parallel pathways

Question 22

What happens if you block Cdc-28?

Answer 22

It blocks everything
• Budding
• DNA replication
• Spindle pole body duplication

Question 23

What happens if you put Cdc-24 to the non-permissive temperature?

Answer 23

It blocks budding

Question 24

What happens if you put Cdc-7 to the non-permissive temperature?

Answer 24

It blocks DNA replication

Question 25

What happens if you put Cdc-31 to the non-permissive temperature?

Answer 25

It blocks spindle body pole formation

Question 26

What controls budding?

Answer 26

Cdc-24

Question 27

What controls DNA replication?

Answer 27

Cdc-7

Question 28

What controls spindle body pole formation?

Answer 28

Cdc-31