Name_____________________

 

                                                                                         TA_______________________

 

Third Exam Fall Biology 50    Molecular Biology and Genetics

November 20, 2001

 

 

(10) 1. Progression through the cell cycle depends on the interaction of two types of regulatory proteins: Kinases and cyclins. List the functions of each and describe how they interact with each other to cause cells to move through the cell cycle.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(10) 2. Describe how the spindle checkpoint was discovered. What genes comprise the spindle checkpoint? What is its significance to our understanding cell cycle control and cancer?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(10) 3. What features are essential for the faithful propagation of a eukaryotic chromosome? Briefly describe how these were discovered.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(10) 4. Yeast cells are either mating type a or mating type α. Using a diagram, illustrate how a single cell can switch mating type. How did this discovery contribute to understanding heterochromatin.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(10) 5. Define oncogenes. Why are most oncogenes expected to be dominant?

 

 

 

 

 

 

 

 

 

 

 

(10) 6. In yeast, the Gal4 protein binds to DNA to activate transcription of GAL7, or GAL10. GAL80 repressses expression by binding to GAL4 protein and preventing it from binding to DNA. In which genes should you be able to isolate galactose constitutive mutations and what characteristics of the protein would the mutation disrupt.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(20) 7. What is the PCR reaction and how does it work. Include a diagram with critical components and their polarity when appropriate (5’ and/or 3’)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(12) 8. A sample of several different human DNAs was cut with the restriction enzyme EcoRI and subjected to gel electrophoresis. A Southern blot was made from the gel and probed with a labeled anonymous cloned sequence. Interpret the data in the blot (in figure) in the following ways.

 

  1. Make a drawing showing how the restriction sites are arranged along the chromosome and indicate the region of the chromosome that must have homology with the probe.
  2. B. How many variant restriction recognition sites are detected in these samples?
  3. What is the genotype of each person whose DNA is represented on the gel?
  4. Are there other genotypes possible that do not occur in this sample of the population?
  5. What patterns would you expect to see in the child if the individual in lane 2 and the individual in lane 4 were to have offspring together? Explain your reasoning.
  6. In fact, the person shown in lane 2 is the offspring of two of the other people whose DNA is shown. Can you identify the parents?

 

       1           2          3          4           5         6          7

 

 

 

 

 

 

 

 

8kb

 

7kb

 

 

 

4kb

 

3kb

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(10) 9.Using a microsatellite DNA probe you have isolated several clones from a library. You would like to identify unique sequences adjacent to these repeat DNA found throughout the genome to establish some microsatellite-based STSs.

  1. How would you determine if there are unique sequences within the clones you have isolated?
  2. The most useful STSs are those associated with microsatellites that are polymorphic in the population (That is, have sequence variants). How could you determine if an STS is associated with a polymorphic microsatellite?

 

 



 

10.  (10 pts.) Below is a drawing of a cloning vector. You want to clone BamH1 digested Kumquat DNA into this plasmid. You cut the plasmid with BamH1, ligate

the Kumquat DNA and the plasmid and transformt he ligation mix into E. coli.

 

 

 

A) Why are the plasmid and the kumquat DNA cut with BamH1?

 

 

B) Why don't you use HindIII instead of BamH1?

 

 

C) Which antibiotic should be incorporated into the media to select transformed bacteria carrying a plasmid?

 

 

D) Which antibiotic should be incorporated into the media for replica plating to identify those E. coli which received a plasmid carrying a Kumquat DNA insert?

 

 

E) In the replica plating experiment, what growth pattern will identify the colony containing a plasmid with a kumquat DNA insert?

 

Honor Pledge:   I have complied with the Honor Code as stated in the Code for Student Conduct.

 ________________

 

ANSWERS

 

1. Progression through the cell cycle depends on the interaction of two types of regulatory proteins: Kinases and cyclins. List the functions of each and describe how they interact with each other to cause cells to move through the cell cycle.

 

Kinase: In presence of ATP will add phosphate to a protein to make phosphorylated protein. Addition of the phosphate either activates or inactivates the target protein. The kinases regulate the transition from G1-S and from G2-M.  The CDK's only work when complexed with cyclin.

 

Cyclin: A protein whose levels change throughout the cell cycle (cyclin), and forms a complex with cdk (cell division kinase, or cyclin dependent kinase). Cyclin binding to CDK is essential for CDK function. There are many cyclins, and each cyclin provides specificity to the CDK for regulating different transitions.

 

 

2. Describe how the spindle checkpoint was discovered. What genes comprise the spindle checkpoint? What is its significance to our understanding cell cycle control and cancer?

 

The spindle checkpoint was discovered by examining the growth of yeast cells following exposure to microtubule damaging agents. Mad1,2 3 and Bub 1,3 mutants continue to divide following microtubule damage. These cells live hard and die young.

It helps us understand cancer because its normal function is to stop the cell cycle if there is damage. In the absence of mad2, damaged cells will continue to proliferate, the damage will accumulate and errors in normal cellular processes will ensue.

 

3. What features are essential for the faithful propagation of a eukaryotic chromosome? Briefly describe how these were discovered

 

Autonomously replicating sequence, Genes (selectable marker, auxotrophic mutant), centromere, telomere

 

Genes were discovered by complementation of auxotrophic mutants

 

ARS’s were discovered by allowing autonomous propagation of plasmids.

ARS plasmids segregate asymmetrically

 

Cen’s were discovered by finding sequences that confer symmetric segregation to ARS plasmids

 

Telomeres are required for the faithful propagation of linear ends of chromosomes.

They were discovered when researchers tried to find sequences that would allow circular plasmids to be maintained as linear plasmids, hence artificial chromosomes

 

 

4. Yeast cells are either mating type a or mating type α. Using a diagram, illustrate how a single cell can switch mating type. How did this discovery contribute to understanding heterochromatin.

 

 

 

 

 

          HML α                                       MATa                                    HMRa

 

Only the genetic information at MAT is expressed. The information at HMR and HML is silenced. Mother cells are able to switch mating type by stimulating a gene conversion type of recombination where the information at MATa switches to MAT α , and vice versa (information at MAT α  switches to MATa). It is a one way exchange of information, thus the silent mating type loci always contain a or α , respectively.

 

Mutations were isolated in which the silenced mating type loci were expressed (sir mutants, silent information repressed). These genes have been shown to be involved in heterochromatin formation, and are used in many eukaryotic cells to facilitate chromosome compaction.

 

5. Define oncogene suppressor genes. Why are most oncogenes expected to be dominant?

 

An oncogene is a gene whose presence predisposes a cell to malignancy. They are typically dominant because the mutations exhibit an abnormal activity (like RAS, locked in the Activating form) or excessive amount of protein.

 

 

6.  

 

Answer: A GAL80 mutation in which the protein is not made or is made but cannot bind to GAL4 would prevent repression and lead to constitutive synthesis. A GAL4 mutation in the portion of the protein to which Gal80 normally binds to repress Gal4 action should be constitutive (A site mutation occurring in the sequence to which Gal4 binds should also lead to constitutive synthesis)

 

7. What is the PCR reaction and how does it work. Include a diagram with critical components and their polarity when appropriate (5’ and/or 3’)

 

PCR polymerase chain reaction.

 

Requirements:

Double strand DNA template

Two oligonucleotides

TAQ polymerase

Nucleotide precursors

 

  1. Denature genomic DNA
  2. anneal oligonucleotides
  3. elongation of primers
  4. denature and repeat steps 2-4 22-32 times.
  5. analyze product by gel electrophoresis

5’ _______________________________________3’

                                                                   3’______5’                                                   

  5’_____3’

3’__________________________________________5’

 

8. Answer: There are four alleles of the RFLP seen.

a) Map

 

RI______RI____________RI_______________RI

      1.0                  3.0                          4.0

 

b) There are two variant restriction sites in the population: the second and third sites on the map above

c) 1: homozygous for 8kb

2: homozygous for 3 + 4

3: heterozygous for 7, 3+4

4:heterozygous for 7,8

5: heterozygous for 8, 3+4

6: homozygous for 7

7: homozygous for 4

d) heterozygous for 4,7:  4, 8:  4, 3+4:  7, 4+3

e) 2 could contribute only 3+4, individual 4 could contribute either 8 or 7. There are therefore 2 possible genotypes in the offspring: 8: 4+3: 7, 4+3

f) 2 got the 4+3 allele from each parent. Therefore the parents must be individuals 3 and 5, the only individuals having that allele of the RFLP.

 

9. The clones you have isolated will contain microsatellite DNA sequences since that was the assay used to identify them. Do they contain unique sequences also? To determine this, you would have to cut the clone into small fragments, reclone the subfragments and use each as a probe in a hybridization with genomic DNA. If only a single band was seen after hybridization with one of these subfragments, you would have identified unique sequence within your clones.

b) To determine if the microsatellite associated with as STS is polymorphic in the population, the oligonucleotides used to detect the STSs surrounding a microsatellite should be used to amplify the DNA from a large sample of individuals in the populations.

 

10. a) compatible ends for cloning

b) HindIII will destroy both selectable markers

c) Amp

d)tet

e)amp resist, tet sens.