Exam 4 Lesson 36

Question 1

How do we study synchronous cells?

Answer 1

We can find natural synchrony or induce it.

Question 2

How do we study naturally synchronous cells?

Answer 2

We can look at them naturally in the early embryo stage, when it all is synchronous, like Xenopus.

Question 3

How do we induce synchrony to study cells?

Answer 3

We can a. add inhibitors and cause cell arrest, b. Remove mitogens, or c. limit nutrients

Question 4

What could we inhibit to make cells synchronous?

Answer 4

DNA synthesis, microtubules, etc.

Question 5

In what type of cells would we remove mitogens? What do mitogens do?

Answer 5

In animal cells. They promote growth.

Question 6

In what type of cell could we limit nutrients to induce synchrony?

Answer 6

Yeast

Question 7

How do we study asynchronous cells?

Answer 7

We can study them a. in situ or b. isolated to analyze at specific phases

Question 8

How do we study asynchronous cells in situ?

Answer 8

Use BrdU

Question 9

How do we isolate asynchronous cells to analyze them at specific phases?

Answer 9

We can isolate them on the basis of a. cell size/shape, b. add fluorescent DNA dye, c. express fluorescent proteins that are cell-cycle regulated (which tells you which phase cell is in)

Question 10

What tools can we use to isolate specific cells?

Answer 10

Flow cytometry or fluorescent activated cell sorting (FACS)

Question 11

What purpose does flow cytometry serve?

Answer 11

Analysis only. Analyzes cell size and properties and takes quantitative data.

Question 12

What purpose does FACS serve?

Answer 12

Analysis and collection

Question 13

How does one study cells in tissue?

Answer 13

Break down cell wall (if plant) with enzymes (in humans, collagenase) FACS+RNA-seq+ proteomics

Question 14

In what cell cycle phase will one see the largest number of cells with the least DNA percentage?

Answer 14

G1

Question 15

What regulates cell cycle in yeast (3)? What are they?

Answer 15

Cdc2ts – mitotic promoter and kinase, cdc25ts – mitotic promoter and phosphatase, wee1ts – mitotic inhibitor and kinase

Question 16

In frog/clam embryos, what was done? Describe experiment.

Answer 16

Preincubate embryos. With 35S methionine (building block for protein), fertilized , SDS page, discovered cyclin, a protein

Question 17

What is MPF?

Answer 17

Maturation promotion factor

Question 18

What was discovered about MPF when the cytoplasm liquid and mitotic extract was centrifuged?

Answer 18

MPF is made up of two proteins. Cyclin and kinase. Kinase is cdc2, the same protein that was encoded in yeast.

Question 19

What kind of kinase is cdc2?

Answer 19

It is a cdk, or cyclin dependent kinase.

Question 20

What is the role cyclin dependent kinases?

Answer 20

They are at the heart of cell cycle regulation. All eukaryotic cells have cdks.

Question 21

How is cell cycle controlled?

Answer 21

Via cyclin dependent kinases

Question 22

Are cyclin dependent kinases always present?

Answer 22

Yes, but not always active

Question 23

How are cyclin dependent kinases controlled/activated?

Answer 23

By cyclin binding, phosphorylation status

Question 24

How do we change phosphorylation status of cdk?

Answer 24

a. cyclin activating kinase activates cdks before or after binding cyclin by phosphorylating T-loop. B. phosphatases – like cdc25 for m-cdk, remove inhibitory phosphates.

Question 25

What proteins do cdks phosphorylate?

Answer 25

ATP substrate

Question 26

Let’s look at the major cyclins. What happens to cyclins during each cell cycle?

Answer 26

They undergo synthesis and degradation.

Question 27

What are the four classes of cyclins?

Answer 27

G1/S-cyclin, S-cyclin, M-cyclin, G1-cyclin

Question 28

G1/S-cyclins

Answer 28

Activates Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase.

Question 29

S-cyclins

Answer 29

Bind Cdks soon after progression through Start and help stimulate chromosome duplications. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of the early events.

Question 30

M-cyclins

Answer 30

Activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis.

Question 31

G1 cyclins

Answer 31

In most cells, help govern the activities of the G1/S-cyclins, which control progression through Start in late G1.

Question 32

What are the major cyclin-Cdk complexes of vertebrates and budding yeast?

Answer 32

G1-Cdk, G1/S-cdk, S-cdk, M-cdk

Question 33

Cyclin and Cdk partner in vertebrate G1-cdk

Answer 33

Cyclin D, cdk4 and cdk6

Question 34

Cyclin and cdk partner in vertebrate g1/S-cdk

Answer 34

Cyclin E and Cdk2

Question 35

Cyclin and cdk partner in vertebrate s-cdk

Answer 35

Cyclin A and cdk2, cdk1

Question 36

Cyclin and cdk partner in vertebrate m-cdk

Answer 36

Cyclin B and cdk1

Question 37

What are the three D cyclins in mammals?

Answer 37

D1, D2, and D3

Question 38

Cyclin and Cdk partner in b.yeast G1-cdk

Answer 38

Cln3, cdk1

Question 39

Cyclin and cdk partner in b.yeast g1/S-cdk

Answer 39

Cln1,2 and cdk1

Question 40

Cyclin and cdk partner in b.yeast s-cdk

Answer 40

Clb5, 6 and cdk1

Question 41

Cyclin and cdk partner in b.yeast m-cdk

Answer 41

Clb1, 2, 3, 4 and cdk1

Question 42

Do concentrations of cdks change during cell cycle?

Answer 42

No

Question 43

If we want to find cells in early G1, what protein would we look for?

Answer 43

Vertebrates – cyclin D, yeast – cln3

Question 44

If we want to find cells in late G1, what protein do we look for?

Answer 44

Vertebrates – cyclin E, yeast – cln 1,2

Question 45

If we want to find cells in S, what protein do we look for?

Answer 45

Vertebrates – cyclin A, yeast, clb5,6

Question 46

If we want to find cells in M, what protein do we look for?

Answer 46

Vertebrates – cyclin B, yeast, clb 1,2,3,4

Question 47

Let’s talk about the activation of a cdk with cyclin. What does cdk have that inhibits it?

Answer 47

Inhibitor phosphate

Question 48

Suppose you add cyclin to cdk with inhibitor phosphate. What happens?

Answer 48

Cyclin binds, but phosphate is still on, so it is inactive.

Question 49

Suppose you add CAK to cdk with cyclin bound. What happens?

Answer 49

CAK will phosphorylate mitogen promoter and dephosphorylate mitogen inhibitor. The cdk exchanges one phosphate for another and is active.

Question 50

How does one turn off cdk once it has been activated?

Answer 50

Add polyubiquitin to cyclin via ubiquitin ligase and send cyclin to lysosome. Cyclin leaves cdk, which then is turned off.

Question 51

We are looking at a graph with the following legend. X is relative amount of DNA per cell and y is number of cells. The dot is high on Y axis but not far on x axis. At what point of cell cycle are we?

Answer 51

We have a lot of cells but not a lot of relative amount of DNA per cell. Cells have just divided, so there is a lot of them, and DNA is split evenly among them, so there is little DNA relative to cell. We are at G1.

Question 52

How much DNA is in each cell per G2 or M phase?

Answer 52

Twice as much as in G1 because cells have replicated but not divided.

Question 53

The cells that have an intermediate amount of DNA are in what phase?

Answer 53

S phase

Question 54

When a cdk is inactive, what blocks the active site?

Answer 54

The t-loop

Question 55

How can we get t-loop to stop blocking cdk active site?

Answer 55

Bind cyclin to it. It moves the t-loop and partially activates cdk.

Question 56

How does t-loop react when CAK binds?

Answer 56

Its conformation changes so enzyme can better bind to its protein substrate.

Question 57

How do Wee1 kinase and cdc25 phosphatase work together to activate and inactivate cdk?

Answer 57

Active cyclin-cdk complex turned off when wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by phosphatase cdc25 activates the cyclin-cdk complex.

Question 58

How does APC/C control proteolysis?

Answer 58

In mitosis, APC/C is activated by association with cdc20, which recognizes specific amino acid sequences on M-cyclin and other target proteins. With E1 and E2 proteins, APC/C assembles polybiquitin chains on the target protein. The polyubiquitylated target is then recognized and degraded by proteasome.

Question 59

How does SCF control proteolysis?

Answer 59

The activity of the uniquitin ligase SCF depends on substrate-binding subunits called F-box proteins, of which there are many different types. The phosphorylation of a target protein allows the target to be recognized by a specific F-box subunit.