Question 1

Which of the following events occurs in mitotic metaphase?
A) Nuclear envelope breakdown
B) Nuclear envelope reassembly
C) Chromosome attachment to spindle microtubules for the first time
D) Chromosome alignment at the spindle equator
E) Mitotic spindle assembly

Accepted Answer

The alignment of chromosomes at the cell equator is the hallmark of metaphase.

Question 2

Consider two mammalian cells, one in G₁ and the other in G₀ (stationary) phase. If they are stimulated to pass the restriction point by the addition of an extracellular proliferation signal, but the signal is then immediately removed, what would you expect to happen?
A) Both cells will replicate their DNA.
B) Only the G₁ cell will replicate its DNA.
C) Only the G₀ cell will replicate its DNA.
D) Only the G₁ cell will start to replicate its DNA, but will stop halfway through the replication and will not reach G₂.
E) Neither of the cells will replicate their DNA.

Accepted Answer

Once a cell passes the restriction point, it is committed to DNA replication (and cell division) even if the stimulating signals are removed.

Question 3

How is centrosome duplication similar to DNA replication?
A) They both use a semiconservative mechanism.
B) They are initiated at around the same time in the cell cycle, near the G₁/S transition.
C) They are both controlled in such a way that they replicate once and only once per cell cycle.
D) They are both separated from their sister copies in mitosis.
E) All of the above.

Accepted Answer

The centrosomes, like the chromosomes, are duplicated only once during each cell cycle. Centrosome duplication is triggered by the activation of G₁/S-Cdk. Both centrosome duplication and DNA replication employ a semiconservative mechanism. The two new centrosomes remain together on one side of the cell until entry into mitosis.

Question 4

You have been studying the effect of loss-of-function mutations in the Cdk inhibitor protein (CKI) p21. You add the drug fucoxanthin to cell cultures harboring either wild-type or mutant versions of the p21 gene. Fucoxanthin is known to induce cell-cycle arrest in G1. After a day, you add the thymidine analog BrdU to the culture media, collect the cells after an hour, treat them with anti-BrdU antibody and the fluorescent DNA stain DAPI, and finally subject them to fluorescence-activated cell sorting (FACS). The FACS data can be viewed as a two-dimensional dot plot composed of thousands of dots, in which each cell is represented by one dot at coordinates that correspond to the intensities of the DAPI fluorescence signal (X axis) and BrdU fluorescence signal (Y axis) for that cell. Answer the following question(s) according to the simplified dot plot below, generated from your experiment.
 
-Which one of the FACS plots (a or b) would you expect to correspond to the loss-of-function p21 mutants? Write down a or b as your answer.

Accepted Answer

Given the significant difference between the results from wild-type and p21 mutant cells, fucoxanthin appears to arrest the cell cycle (in G?) by activating a p21-dependent pathway. Loss of p21 (plot a) therefore blocks the cell-cycle arrest induced by the drug.

Question 5

Which of the time points (A to E) in the following schematic drawing of the mammalian cell cycle represents the restriction point?

Accepted Answer

Under favorable conditions and in the presence of signals to grow and divide, cells in early G₁ (or G₀) progress through a commitment point near the end of G₁ known as Start (in yeasts) or the restriction point (in mammalian cells).

Question 6

Consider two kinesin motor proteins at the mitotic spindle midzone: kinesin-5 is a tetrameric motor that walks toward the plus end of both microtubules to which it is attached via its motor domains; kinesin-14, on the other hand, walks toward the minus end of one microtubule while it is attached to another microtubule via its tail domain. How do these motors affect the length of the spindle?
A) They both work to shorten the spindle.
B) Kinesin-5 works to shorten the spindle whereas kinesin-14 works to lengthen it.
C) Kinesin-5 works to lengthen the spindle whereas kinesin-14 works to shorten it.
D) They both work to lengthen the spindle.

Accepted Answer

While kinesin-5 pushes the two poles of the spindle apart by walking toward the plus end of antiparallel microtubules, kinesin-14 pulls the two poles toward each other by walking toward the minus end of interpolar microtubules in the midzone.

Question 7

In the fission yeast Schizosaccharomyces pombe, the proteins Cut1 and Cut2 form a complex that is catalytically inactive. At the onset of anaphase, Cut2 is polyubiquitylated by a large E3 complex containing Cut4, Cut9, Cut23, and other proteins, and is subsequently destroyed. Cut1 then cleaves Rad21, a non-SMC component of a complex that also contains two SMC proteins, thus allowing sister-chromatid separation. Mutations in the cut genes lead to the cut phenotype, in which the cell attempts cytokinesis without chromosome segregation. Indicate which of the following proteins or protein complexes corresponds to or contains the product of the genes cut1 (A), cut2 (B), cut4 (C), and rad21 (D). Your answer would be a four-letter string composed of letters A to D only, e.g. DCAB.
( )	Securin
( )	Cohesin
( )	Anaphase-promoting complex
( )	Separase

Accepted Answer

Rad21 is a Scc1 homolog, which is cleaved by separase in the metaphase-anaphase transition.

Question 8

Indicate whether each of the following descriptions better applies to a Wee1 protein (W) or a Cdc25 protein (C). Your answer would be a four-letter string composed of letters W and C only, e.g. WWCW.
( )	It is a protein kinase.
( )	It activates M-Cdk complexes.
( )	It is activated by M-Cdk complexes.
( )	Its loss in fission yeast results in small cell size.

Accepted Answer

Wee1 kinases phosphorylate and inactivate cyclin-Cdk complexes; Cdc25 phosphatases do the opposite. In a positive feedback loop at the onset of mitosis, M-Cdk inactivates its inactivator, Wee1, and activates its activator, a Cdc25. The small cell size in wee mutants can result from Wee1 depletion.

Question 9

Which one of the following chromosomes has formed stable attachment(s) to the spindle microtubules? 
A) 
B) 
C) 
D) 
E)

Accepted Answer

Bi-oriented chromosomes are stabilized due to the unique tension created.

Question 10

Resveratrol is a natural compound found in red grapes (and red wine) and is thought to have beneficial effects in mammals, such as preventing tumor growth and delaying age-related diseases. In vitro, resveratrol and its derivatives have been shown to cause cell-cycle arrest in S phase and induce apoptosis. You have analyzed the DNA content of cultured cells in the presence and absence of these drugs using fluorescence-activated cell sorting. Compared with control cells (dashed line), which of the following curves do you think better represents the DNA content of cells treated with these compounds?

Accepted Answer

S-phase arrest will increase the proportion of cells in this phase; that is, cells with a relative cellular DNA content of between 1 and 2.

Question 11

The cell cycle can be arrested in mitosis when a single sister-chromatid pair is mono-oriented on the mitotic spindle. If at this point a glass microneedle is used to pull the mono-oriented chromosome toward the pole to which it is not attached, the cell proceeds to anaphase. This observation confirms that …
A) mono-oriented chromosomes are stable.
B) mechanical tension drives the formation of bi-oriented chromosomes.
C) bi-oriented chromosomes activate the spindle assembly checkpoint.
D) lack of mechanical tension at the kinetochore in at least one chromosome prevents entry into anaphase.
E) mechanical tension at the kinetochore in at least one chromosome is required for entry into anaphase.

Accepted Answer

This important experiment confirms the importance of mechanical tension within the kinetochores in the spindle assembly checkpoint mechanism.

Question 12

Which of the following is more directly driven by active M-Cdk?
A) Centrosome maturation
B) Centrosome duplication
C) Nuclear envelope reassembly
D) Inactivation of APC/C
E) Cell cleavage

Accepted Answer

The M-Cdk is responsible for most events in early mitosis, including spindle assembly and centrosome maturation. It is inactivated later in mitosis, resulting in the events of telophase and cytokinesis.

Question 13

Mammalian Cdk inhibitor proteins (CKIs) can be grouped into two families based on their structural and functional differences. The Cip/Kip family proteins (e.g. p21) have a broad binding specificity. These proteins bind preferentially to already formed cyclin-Cdk complexes and thus enhance complex formation. However, they inhibit the kinase activity of most complexes (e.g. S-Cdks), except in the case of G?-Cdk complexes where no inhibition occurs. Consequently, Cip/Kip family proteins have an overall positive effect on Cdk4/6 activity due to their help in bringing the subunits together. In contrast, the inhibitors of the INK4 family (e.g. p16) bind only to the Cdk subunit of G?-Cdks and prevent binding of both the G? cyclins and the Cip/Kip family CKIs. Based solely on these findings, would you expect p16 to activate (A) or inactivate (I) the S-Cdks in the presence of limited amounts of p21?

Accepted Answer

In the absence of p16, p21 is sequestered by the G?-Cdk complex. Active p16, however, prevents p21 from binding, making it available to inhibit the S-Cdk complexes.

Question 14

Which of the following simplified diagrams better shows the changes in the concentrations of three major cyclin-Cdk complexes (G?/S-Cdk, S-Cdk, and M-Cdk) in the cell in different stages of the cell cycle?

Accepted Answer

The G₁/S-Cdk activity (solid black curve) peaks in late G₁ and falls in S phase. The S-Cdk levels (solid gray curve) remain elevated through S and G₁ until early M phase. M-Cdk (dashed gray curve) accumulates before entry into mitosis, but their level falls in mid-mitosis.

Question 15

Indicate whether each of the following occurs mainly in G₁ phase (G), S phase (S), or G₂ phase (H) of the cell cycle. Your answer would be a four-letter string composed of letters G, S, and H only, e.g. HGSS. ( ) DNA helicase activation ( ) DNA helicase deposition on DNA at the replication origins ( ) ORC phosphorylation ( ) Licensing of replication origins

Accepted Answer

The loading of inactive DNA helicases onto the replication origins and the formation of the prereplicative complex (preRC) occur in late mitosis and early G₁. In S phase, the helicases are activated to initiate DNA replication, while the origin is prevented from refiring by origin recognition complex (ORC) phosphorylation and other mechanisms.

Question 16

Fill in the blank: "The … is a large structure formed at the centromeric region of each eukaryotic chromosome. It captures spindle microtubules in mitosis, and therefore serves to attach the chromosomes to the spindle poles."

Accepted Answer

Kinetochores are giant protein structures that attach sister chromatids to the mitotic spindle.

Question 17

Sort the following schematic diagrams (A to F) to reflect the order of events in a typical eukaryotic M phase. An interphase cell in G₂phase is drawn on the left for comparison. Your answer would be a six-letter string composed of letters A to F only, e.g. BCEADF.

Accepted Answer

The M phase includes mitotic prophase (C), prometaphase (F), metaphase (E), anaphase (D), telophase (A), and finally cytokinesis (leading to B).

Question 18

You have been studying the effect of loss-of-function mutations in the Cdk inhibitor protein (CKI) p21. You add the drug fucoxanthin to cell cultures harboring either wild-type or mutant versions of the p21 gene. Fucoxanthin is known to induce cell-cycle arrest in G1. After a day, you add the thymidine analog BrdU to the culture media, collect the cells after an hour, treat them with anti-BrdU antibody and the fluorescent DNA stain DAPI, and finally subject them to fluorescence-activated cell sorting (FACS). The FACS data can be viewed as a two-dimensional dot plot composed of thousands of dots, in which each cell is represented by one dot at coordinates that correspond to the intensities of the DAPI fluorescence signal (X axis) and BrdU fluorescence signal (Y axis) for that cell. Answer the following question(s) according to the simplified dot plot below, generated from your experiment. -Indicate which boxed region (1, 2, or 3) in the FACS plots corresponds better to each of the following phases of the cell cycle. Your answer would be a three-digit number composed of numbers 1 to 3, with each number used once, e.g. 312. ( ) G₁ phase ( ) S phase ( ) G₂ and M phases

Accepted Answer

After a short incubation with BrdU, cells that are replicating their DNA (in S phase) would incorporate the compound and would show a high BrdU signal compared to cells that are in G₁, G₂, or M phases. The DAPI signal intensity represents the total DNA content, which increases from G₁ to S to G₂ phases.

Question 19

Indicate true (T) and false (F) statements below regarding mitosis and the changes that it brings about compared to interphase. Your answer would be a four-letter string composed of letters T and F only, e.g. TFTF.
( )	Microtubules become greatly stabilized in mitosis compared to interphase.
( )	The number of ?-tubulin ring complexes in the centrosomes increases greatly during mitosis compared to interphase.
( )	Chromosomes stabilize mitotic microtubules through activation of Ran monomeric G protein.
( )	A bipolar spindle can be formed even in the absence of centrosomes.

Accepted Answer

Mitosis is accompanied by a dramatic reorganization of the microtubule cytoskeleton, with a significant increase in microtubule instability. At the same time, centrosome maturation greatly increases microtubule nucleation. The capture of microtubules by chromosomes stabilizes microtubules in part by activating Ran, a monomeric GTPase. The ability of mitotic chromosomes to stabilize and organize microtubules makes it possible to form bipolar spindles even in the absence of centrosomes.

Question 20

The following schematic diagram shows the activation of M-Cdk in mitosis. Indicate which proteins below correspond to those indicated as A to D in the diagram. Your answer would be a four-letter string composed of letters A to D only, e.g. BACD.
  
( )	Wee1
( )	CAK
( )	Cdc25
( )	M-cyclin

Accepted Answer

Activation of M-Cdk is sharpened by positive feedback loops in which M-Cdk activates its activator (Cdc25) and inactivates its inactivator (Wee1) by phosphorylation.

Quiz 17: The Cell Cycle

Which of the following events occurs in mitotic metaphase?

Consider two mammalian cells, one in G₁ and the other in G₀ (stationary) phase. If they are stimulated to pass the restriction point by the addition of an extracellular proliferation signal, but the signal is then immediately removed, what would you expect to happen?

How is centrosome duplication similar to DNA replication?

Which of the time points (A to E) in the following schematic drawing of the mammalian cell cycle represents the restriction point?

Which one of the following chromosomes has formed stable attachment(s) to the spindle microtubules? A) B) C) D) E)

Which of the following is more directly driven by active M-Cdk?

Which of the following simplified diagrams better shows the changes in the concentrations of three major cyclin-Cdk complexes (G?/S-Cdk, S-Cdk, and M-Cdk) in the cell in different stages of the cell cycle?

Fill in the blank: "The … is a large structure formed at the centromeric region of each eukaryotic chromosome. It captures spindle microtubules in mitosis, and therefore serves to attach the chromosomes to the spindle poles."

Sort the following schematic diagrams (A to F) to reflect the order of events in a typical eukaryotic M phase. An interphase cell in G2phase is drawn on the left for comparison. Your answer would be a six-letter string composed of letters A to F only, e.g. BCEADF.

The following schematic diagram shows the activation of M-Cdk in mitosis. Indicate which proteins below correspond to those indicated as A to D in the diagram. Your answer would be a four-letter string composed of letters A to D only, e.g. BACD. ( ) Wee1 ( ) CAK ( ) Cdc25 ( ) M-cyclin

Sort the following schematic diagrams (A to F) to reflect the order of events in a typical eukaryotic M phase. An interphase cell in G₂phase is drawn on the left for comparison. Your answer would be a six-letter string composed of letters A to F only, e.g. BCEADF.