Molecular Cell biology-Drawing structures

Question 1

Draw the cell undergoing prophase.

Answer 1

Refer to notes, must have centrosomes, mitotic spindle (+ends showing optional), condensing chromosomes, intact nuclear envelope, kinetochores showing. (motor proteins optional) (if the motor proteins are shown, dynein should be associated with cell membrane and pulls on astral MTs, kinesin between interpolar MTs)

Question 2

Draw the cell in prometaphase.

Answer 2

Must show NEB, (fragments of nuclear envelope), centrosomes should be at both poles, mitotic spindle showing, interpolar, astral and some kinetochore MTs. (Show dynein and kinesin 14)

Question 3

Draw the cell in metaphase.

Answer 3

All chromosomes have kinetochore-MT attachments, fragments of nuclear envelope, centrosomes at opposite poles.

Question 4

Draw the cell in anaphase.

Answer 4

Anaphase A. Show shortening of MTs attached to kinetochores and chromosomes moving to opposite poles.
Anaphase B. Show the effects of dynein and kinesin 5. (kinesin 5 force on the interpolar microtubules, interpolar MTs also elongate. Dynein pulls centrosomes to opposite poles.)

Question 5

Draw or just write (as a flow chart) the experiment for the rescue by complementation of S. pombe cdc2-ts. Starting from whole DNA sequence.

Answer 5

Show the steps of DNA library. (DNA –> RNA–> mRNA –> RT –> cDNA –> clone into plasmids –> cDNA library.
From library –> transform plasmids into plated ts-cdc2 mutants grown at restrictive temperature –> plate at restrictive –> one colony survives –> isolate plasmid –> determine sequence and protein.

Question 6

Show the in-vitro kinase (histone H1) assay that they used to determine the cdc28 kinase activity.

Answer 6

IP using anti-cdc28
add radioactive gamma-p32-ATP
add purified histone H1
incubate
SDS-PAGE
radioactive band at 29kDA

Question 7

Draw the MPF activity against location in meiosis chart.

Answer 7

Refer to notes.

Question 8

Draw the activation of any type of cdk.

Answer 8

inactive –> binding to cyclin partner (partially active) –> phosphorylation of T-loop –> CKI

Question 9

Draw the activation process of APC-cdc20 and its effect on the M-cdk. (Do not show mad2 and SAC)

Answer 9

refer to notes.

Question 10

Draw the regulation of mitotic onset.

Answer 10

refer to notes, (cdk from inactive to active with positive feedback loops)

Question 11

Draw the mitotic spindle, including motor proteins and all types of MTs.

Answer 11

refer to notes

Question 12

Draw the general process of kinetochore capture.

Answer 12

Refer to notes

Question 13

Draw the different kinetochore-MT attachements. Also show the activity of aurora B.

Answer 13

refer to notes

Question 14

Draw the pathway for the destruction of the cohesin complex.

Answer 14

Refer to notes.

Question 15

FISH experiment.

1. How many dots for wt?
2. How many dots for APC mutant?
3. How many dots for securin mutant?
4. How many dots for APC/securin double mutant.
5. Separase mutant.
6. For all of these, if scc1 was labeled, where would it show up?

Answer 15

Refer to notes

Question 16

Draw the SAC. (only one chromosome needs to be shown, include both APC and cdk1)

Answer 16

Kiss and run model.

Question 17

Draw the RhoA activation leading to assembly of contractile ring.

Answer 17

refer to notes.

Question 18

Draw a diagram showing the localization of actin, MHC (myosin heavy chain), RhoA, ECT2, CYK4

Answer 18

refer to notes.