BIOL 377 Phosphatases

Name: [name here]

 

1.) Tell me the limit of resolution of the following the microscopy techniques.  You may use an answer more than once.  Options are as follows: a.) ~ 0.1nm, b.) 30-50nm, c.) ~ 200nm   (5pts)

 

1a.)  electron microscopy – [~ 0.1nm]

 

1b.) super resolution microscopy – [30-50nm]

 

1c.) confocal microscopy – [~ 200nm]

 

1d.) transmitted light microscopy – [~ 200nm]

 

1e.) fluorescence microscopy – [~ 200nm]

 

2.)  S. cerevisiae, A. thaliana, E. coli, C. elegans, M. musculus and D. melanogaster are all examples of model organisms.  List three traits of model organisms that we discussed in class.  3pts

• They are easy to manipulate
• They have a short life span or life cycle hence mature rapidly
• They are very easy to observe and understand; isolating mutant organisms is therefore easy.

 

3.) You want to observe the behavior of a GFP-labelled protein in a live tobacco BY-2 cells.  How do you do this? 5pts

 

The GFP protein is fused to the live tobacco BY-2 cells. A sample of it is put on a slide under a fluorescence microscope where a light source emits multiple wavelengths. In the microscope, the first filter allows one wavelength to pass through, blue light 488nm that is reflected by a mirror through the objective lens into the sample. In the tobacco cells, the exited fluorophore emits green light that is collected through the objective lens and back through the mirror then through a second filter that allows only the emission of light that is 515 nm wavelength which passes to the eye piece directly for observation or to a camera. That is how the GFP is used to observe live tobacco BY-2 cells

 

4.) Green fluorescent protein can be expressed as a chimeric protein.  This means that GFP can be __can be fused to any protein of interest i.e. any particular protein to be studied hence allowing scientists to look at protein dynamics in living cells__.  5pts

 

5.) Kinases are a type of protein that are very important in cell cycle regulation and cell signalling.  What do kinases do? 2pts

 

Kinases are enzymes that activate specific amino acids by phosphorylating them using a phosphate from adenosine triphosphate hence influencing metabolism.

 

6.) Phosphatases are a type of protein that are also involved in cell cycle regulation and cell signalling.  What do phosphatases do? 2pts

 

Phosphatases are enzymes which split phosphor-ester bonds in organic substrates that have phosphate moieties liberating the phosphate moiety in the presence of water.

 

7.) Ethylene is a hormone involved in fruit ripening.  We looked at this hormone in the context of fruit ripening in tomatoes.  Plant biologists knocked down the expression of the ACC synthase using antisense (essentially RNAi) in transgenic tomatoes.  The transgenic ACC synthase knock down tomatoes were unable to ripen.  What were the researchers able to conclude from this experiment?  5pts

In introducing an antisense into the enzyme ACC synthase, ethylene synthesis was interfered with. Thus the method proved to be useful in preventing ripening as ethylene is necessary for ripening. The conclusion is that ripening is not spontaneous and that it requires ethylene to proceed. Hence introduction of antisense constructs into ACC synthase is an effective way to limit ethylene production and control ripening.

 

8.) ABA is a plant hormone that binds to the PYL1 receptor.  This when ABA is bound to PYL1 it is able to bind to the protein PP2C.  This binding inhibits the function of PP2C.  What is the function of PP2C?  5pts

Protein phosphatase type 2 C inhibits SnRK2s (Sucrose non-fermenting 1- related protein kinases) which are normally activated by abscisic acid.

PP2C also inhibits SnRK2.4 a kinase independent of abscisic acid.

It also plays a role in regulating the growth of roots in response to salinity in conjunction with SnRK2.4 protein and ABI1 protein (Abelson interactor 1).

The inactivation of PP2C permits SnRK protein kinases (among other proteins) to phosphorylate and activate ABA-inducible TFs, promoting transcription of ABA inducible genes; this further supports the inhibitory action of PP2C.

 

9.) Long form experimental question

(15 pts). 

Pidge, the resident scientist of team Voltron, is examining the mysterious space spores that they found outside while repairing the ship.  After collecting a few spores in space, she brings them into the lab to further examine them.  She is curious to determine how the spores are able to exhibit bioluminescence.  Pidge separates the spores into two different treatments: light and dark.  When spores are stored in the light, they do not glow; while in the dark, the spores glow.  This indicates that spore luminesce is linked to light levels that the spores are exposed to.

 

She decides what she wants to take samples from the spores and performs the following analyses.  Pidge collects genomic DNA and Pidge is able to assemble a whole genome sequence.

 

Pidge collects RNA from two types of spore samples: luminescing (glowing) and non-luminescing (non-glowing) spores. Her glowing spores were in the dark treatment and her non-glowing spores were in the light treatment.  She generates transcriptomes from the spore treatments and is confident that she has captured all of the genes that are expressed in each treatment.

 

Pidge has identified candidate genes that are involved in spore luminesce, from her transcriptomes.  She has named the candidates GLOW1, GLOW2, GLOW3 and GLOW4.

 

GLOW1 encodes a putative protein that looks similar to luciferase (the protein that allows lighting bugs butts to glow).  GLOW1 has several serine resides that could be phosphorylated.  GLOW1 is expressed in dark spores.

 

GLOW2 encodes a putative protein kinase.  The protein kinase has a domain that allows for it to interact with the GLOW1.  GLOW2 is expressed in dark spores.

 

GLOW3 encodes a MADS-box transcription factor.  GLOW3 is expressed in dark spores.

 

GLOW4 encodes a putative phosphatase. The GLOW4 has a domain that allows for it to interact with GLOW1. GLOW4 is expressed in light spores.

 

Experiment 1.) With the information from when these genes are expressed, light vs dark, as well as what type of protein they encode for, Pidge draws a pathway with the that each of these proteins act in the process of glowing.  There is more than one “correct” answer for this, I will accept all answers that biologically could make sense.

 

Question 9a.) Write out/describe a pathway for the that each of the GLOW proteins could be acting in this pathway 4pts.

 

Example pathway:

Dark spores:  —–>  glowX  -> glowY -> glowC etc.    spores moved to light -> glowE.

 

Start with spores that were just placed in the dark.  Write out the linear path of each gene and what the protein would have activity would be in the dark and then what would happen in the light.  Extra credit awarded for a description of what each protein does to the next protein in the pathway such as phosphorylates, ubiquitinates, dephosphorylates etc.

 

Start with

Dark spores ——-> Glow 3(MADS box transcription factor)à Glow 2 (encodes for protein kinases that phosphorylate serene residues)à Glow 1(luciferase like molecule that fluoresce) when moved to light àGlow 4(phosphatase breaks down the phosphate-ester bonds to end fluorescence)

 

End with

Light spores ——–> Glow 4 (phosphatase cleaves phosphate off serine residues in presence of light)àGlow 1(serine residues do not fluoresce)

 

 

Experiment 2.)  Pidge is able to clone each GLOW gene and express these proteins in yeast.  She decides to test the function of the genes in the pathway based on their protein function in vitro.  Pidge has generated a primary antibody to GLOW1.

 

Pidge wants to demonstrate that the GLOW1 protein and physically interact with the GLOW2 and GLOW4 proteins.  Question 9b.) What in vitro test can she perform? (3pts)

 

She can perform a tandem affinity purification test

 

 

 

 

Experiment 3.) From the previous experiment, Pidge has proven that GLOW1 can physically interact with GLOW2 and GLOW4.  Pidge then hypothesizes that GLOW1 is phosphorylated and activated by GLOW2 and that it is dephosphorylated by GLOW4.

 

She designs an experiment to test the activity of GLOW1 in vivo in the yeast.

 

Question 9c.) What type of microscopy would Pidge use to test if GLOW1 is able to fluoresce or not? (2pts)

 

Fluorescence microscopy.

 

 

Question 9d.) Tell me which of these protein combinations would fluoresce or not. (4pts)

 

 

GLOW1 + GLOW2 = [This would fluoresce]

 

GLOW1 + GLOW4 = [No fluorescence]

 

GLOW2 + GLOW 4 = [No fluorescence]

 

 

 

Experiment 4) Pidge then wants to determine which serine residues are phosphorylated in GLOW1 by GLOW2.  Question 9e.) How can Pidge test if these resides are necessary for phosphorylation? (2pts)

 

The only way she can test if the residues are necessary for phosphorylation is by knocking out the genes encoding for them. Exclusion of the residues is the surest way of examining their importance and necessity.

 

10.) We studied the role of the MPF complex in class and its role in cell cycle regulation.  What does the MPF complex control? 3pts

 

It controls cell entry into mitosis i.e. it phosphorylates proteins, hence activating these proteins necessary for mitosis thus triggering mitosis.

 

Multiple Choice Questions 11-15.   Nodulation in legumes.  Highlight the correct answer!

 

11.) We looked at two types of mutant screens that were performed in legumes.  Which of the following was NOT one of the screens that we examined in class? 2pts

 

a.) selection of non-nodulating mutants                     b.) selection of hyper-nodulating mutants

 

c.) selection of nodulating mutants

 

12.) The mutant screens revealed the receptors of NFR1 and NFR5 that are found on the surface of the root epidermal cells.  What was the ligand that NFR1 and NFR 5 recognize? 2pts

 

a.) cytokinin                b.) bacterial flagellin e.g. flg 22

 

c.) N-acetylglucosamine polymers e.g. NOD factors d.) bacterial cell walls

 

 

13.) The har1 mutant plants displayed what type of nodulation phenotype? 2pts

 

a.)  hypernodulation                           c.) hyper-infection

 

b.) non-nodulating                              d.) no infection

 

 

14.) The wild-type Hit1 gene encoded a protein that was called LHK1.  What type of protein is LHK1? 2pts

 

a.)  serine-threonine kinase              c.) histidine kinase

 

b.) receptor tyrosine kinase                 d.) calcium dependent kinase

 

 

15.) The hormone cytokinin binds to LHK1.  What organ is formed in the root as a result of the signalling from this hormone? 2pts

 

a.) nodule                    b.) root hairs

 

c.) cortex                     d.) xylem

 

 

Questions 16-18 Matching. Mitosis. 3 pts

 

Match each microtubule population with the function it performs in the mitotic spindle:

a.) support framework and interdigitate with the opposing pair

b.) attach to chromosome at the kintechore

c.) radiate from centrosome, determine cleavage plane

 

16.) astral microtubules – [c. radiate from centrosome, determine cleavage plane]

 

17.) kintechore micotubles -[b. attach to chromosome at the kintechore]

 

18.) polar microtubules –  [a. support framework and interdigitate with the opposing pair]

 

 

Multiple choice questions. Mitosis and cytokinesis. Highlight the correct answer!

 

19.) We covered the function of the mitotic spindle in class.  Are centromeres actually necessary for mitosis in cells?  2pts

 

a.) Yes             b.) No

 

20.) Once the sister chromatids are lined up along the division plane during mitosis in metaphase, and before the separation of the chromatids what type of state are the kintechore microtubules in? 2pts

 

a.) treadmilling                        b.) rapid polymerization

 

c.) rapid depolymerization

 

21.) The purpose of the spindle assembly checkpoint is to? 4pts

 

a.) prevent the transition from prophase to metaphase if necessary

 

b.) prevent the transition from metaphase to anaphase if necessary –ANSWER

 

c.) prevent the transition from anaphase to telophase if necessary

 

d.) prevent the transition from telophase to cytokinesis if necessary

 

22.) Cytokinesis in animal cells and plant cells works by two different processes.  Which of the following statements is correct? 3pts

 

a.) animal cells form a cell plate; plant cells form a cleavage furrow

 

b.) animal cells form a cleavage furrow; plant cells form a cell plate- CORRECT

 

c.) animal cells form a cleavage furrow; plant cells form a plasmodesmata

 

 

Matching 23-25.  Match the event with the correct process, in regard to cell division without mitosis and mitosis without cell division.  You may use an event more than once. 3pts

 

23.) meiosis – [b. cell division without mitosis]

 

24.) plant trichome development – [a. mitosis without cell division]

 

35.) insect embryo development – [a. mitosis without cell division]

 

 

Matching 26-31. Match each event with the purpose of apoptosis.  You can use each rationale more than once. Options:  a.) apoptosis for development   or b.) apoptosis for defense/protection  6pts

 

26.) formation of fingers and toes – [apoptosis for development]

 

27.) loss of a tadpole tail – [apoptosis for development]

 

28.) cells with DNA damage – [apoptosis for defense/protection  ]

 

29.) immune cells at the end of an infection – [apoptosis for defense/protection]

 

30.) neural innervations – [apoptosis for development]

 

31.) cells infected with viruses  – [apoptosis for defense/protection]