Based on this information, what type of macromolecule (be specific) is responsible for the spread of disease in each case? Considering what you know about the central dogma of molecular biology, describe what is surprising about each of these results?

For honours project, you have joined a lab which studies plant diseases.  Your supervisor has as library of novel pathogens capable of causing disease in plants.  While they have conducted some preliminary investigations into these pathogens, you have been recruited to help characterize one of them more fully and begin working towards a treatment for the disease it causes.

Your first task is to select the pathogen you would like to investigate for the coming year.  You have been given the following pieces of information as you consider this choice.

• Neither one of these pathogens is able to grow by itself on a nutritive medium in the absence of plant cells.

• In the initial screening, a sample of each pathogen was treated with: 1) a nuclease that digests all types of nucleic acids, 2) a protease, and a 3) lipase. These treated samples where then inserted directly into plant cells to determine if they were still able to cause an infection. The results of these experiments are summarized in the following table.

• Both pathogens were screened for reverse transcriptase, endonuclease, integrase, and transposase activity. Pathogen A was negative for all of these.  Pathogen B was positive for reverse transcriptase, and integrase activity only.

• The nucleic acids from pathogen B were isolated and sequenced. The results revealed that they were composed of 25% A, 40% U, 20% G, 15 % C.

1. Based on this information, what type of macromolecule (be specific) is responsible for the spread of disease in each case? Considering what you know about the central dogma of molecular biology, describe what is surprising about each of these results? If applicable, describe any atypical transfers of biological information which occurs in each case. (6 marks)

The macromolecule responsible for the transmission of pathogen A is _________________.

The macromolecule responsible for the transmission of pathogen B is _________________.

2. Which one of these pathogens would you personally prefer to study further and why? (1 mark)

Ultimately supervisor decides that it is more important to characterize pathogen B and asks you to pursue this for your honours project.  All questions from this point forward refer to pathogen B.

Looking to gather more information about your pathogen of interest, you read your supervisor’s field notes about its collection.  They describe having collected specimens (from which the pathogen was later isolated) from several diseased blueberry bushes in a large blueberry barren (a large field of blueberry plants).  They noted their surprise that only seemingly random plants scattered throughout the field had been affect by this disease and that most of the neighboring plants remained healthy.

3. To determine if you can replicate this seemingly random infection pattern in the lab, you expose a large number of plant cell cultures with pathogen B. You determine that ~10% of the exposed colonies appear to have been infected by this pathogen (i.e. – there is evidence of unhealthy and dying cells in the culture dishes).  To confirm that your pathogen entered all of the plant cell cultures and ensure that bad laboratory technique isn’t blame for this low infection rate, you conduct targeted sequencing for pathogen B.  To your surprise, the sequencing results reveal that in > 95% of your plant cell cultures, the pathogen’s genome is present and has integrated with the host cell’s genome.  Interestingly the pathogen’s genome has integrated at different locations in each of the infect plant cell’s genomes.

1. Considering what you know about chromosome structure and organization, predict the potential for symptoms of infection if the pathogen’s genome were to be integrated at each of the sites labeled on the Geimsa stained plant chromosome shown below. Explain your rationale in each case, clearly indicating why you would or would not expect to see expression of the pathogen’s genes and therefore disease symptoms. (6 marks)

1. Would your answers in part a change if sequencing results confirmed the presence of a barrier sequence within the terminal repeats at each end of the integrated pathogenic sequence? Why or why not? (2 marks)

2. If the affected plant chromosome is ~ 2.82 cm in its linear form (i.e. – free from proteins), how many base pairs would you estimate the chromosome consists of? (2 marks)

1. pproximately how long would you expect this chromosome to be when it adopts the conformation shown in part a of this question? (2 marks)

4. To understand how this pathogen spreads, you decide to study the replication of its genome following its integration within the host cell’s genome.
5. In one infected specimen you allow replication to proceed in the presence of radioactive phosphates for 1 minute and then terminate replication by breaking open the plant cells and their nuclei. You extract the newly synthesized DNA, heat denatured it (to separate parental and newly synthesized strands), and examine the radioactive DNA. The DNA appeared as is shown in the diagram below.

1. You have determined that in one of your infected plant cell cultures, the pathogen’s genome was inserted into the middle of a gene which is an essential component of the FACT complex abolishing its expression. Describe how you expect that this would affect the process of replication in these cells and why.  (2 marks)

5. In some instances, microbial genomes that have been integrated with a host cell’s genome may also be removed from it as part of their life cycle.
6. Name the process(s) by which the host cell’s DNA would be repaired following excision of the pathogen’s genetic material and briefly describe the process(s). If there is more than one possible repair mechanism that could be used, be sure to describe the circumstances under which each one would be selected.  (6 marks)

(e.g. BER,. NER, mismatch repaire, dsDNA break repair)

1. Describe at least one possible downside to the repair mechanism(s) you have outlined. (2 marks)

6. You are also interested in developing treatments that could be used to rescue plants which are affected by the disease caused by pathogen B.
7. Remembering that during the initial characterization of this pathogen, nucleases were able to eliminate the infectious potential of this substance, you decide to try treating infected plant cells with a nuclease you have on hand in the lab, micrococcal nuclease (MNase). However, when you return to the lab the next day all of the treated cells have died.

Undeterred, you try again using a series of shorter nuclease treatments.  You isolate chromatin from the cells at each time point, dissociate the nucleic acids from the protein components and separate the nucleic acids on a gel.  You obtain the results shown in the adjacent figure.

1. Why did this speic fracifgmentation pattern arise? Why aren’t all of the nucleic acids completely broken down into their component nucleotides after this treatment? (2 marks)

1. What did you not take into account when choosing this treatment that ultimately resulted in the death of these cells? (1 mark)

1. Knowing that some chromatin conformations are more or less permissive to the expression of the pathogen’s genes, you decide to try treating your infected cells with compounds that alter the activity of writer and eraser proteins. Predict the effect of the following compounds on the expression of the pathogen’s genes and therefore disease symptoms.  Be sure to explain your reasoning in detail. (6 marks)
2. Histone deacetylase inhibitor –

1. Histone kinase activator –

1. Propose another treatment approach you would like to explore/develop for this pathogen. Explain why you have selected it and be specific as to why you believe it might be able to treat this plant disease  Keep you answer concise (no more than one paragraph of 5-8 sentences). (3 marks)