GENE20001- Biochemical Pathways Toxic & Harmless With Medicinal Compounds, DNA - Biology Assignment Help

Assignment Task -                 
 

General background information and overview.

The special pretense is a haploid fungus with a life-cycle similar to that of baker’s yeast Saccharomyces cerevisiae. This organism is important for the biotechnological product of rare medicinal compounds. Unfortunately, in some lab cultures, a harmless by-product is often produced (by-product B), and this is readily converted to a toxin (Compound C), via a biochemical pathway involving ‘Enzyme P’. An alternate pathway exists for the breakdown of by-product B into a harmless compound, however, the enzyme that catalyzes this reaction is not very active. These two biochemical pathways are shown below.

A gene called reps has previously been found to be involved in the biosynthesis of Compound C where it acts as a negative regulator of the Enzo gene. The rep gene encodes a C6 zinc cluster transcription factor called ‘Repressor X’ which has previously been isolated and characterized. 

A new research lab is investigating a number of strategies to try to reduce the production of the toxic compound C

1. They want to introduce specific mutations to increase the activity of rep and are wanting to predict how different mutants may affect the RepX protein. (Section 1)

2. A related species, Special hypothetical, does not appear to produce Compound C under laboratory conditions. They have cloned and sequenced a molecule of DNA that may contain a gene related to reps, but they are unsure of the sequence annotation. (Section 2)

3. They have found two additional genes, rep and reps, in S. pretense that also appear to regulate Compound C production and they want to determine how these interact with repX. (Section 3)

4.  They wish to explore how they may be able to increase the expression of Enzyme W so that by-product B is broken down via the alternate pathway. This will in turn reduce the amount of by-product B that can be converted to Compound C. (Section 4)

Instructions for completing the assignment

• All of the relevant sequences and data for each section are provided at the end of the document. 

• In each section, you are provided with the relevant information and referred to the appropriate data to complete the analysis. 

• All of the questions are to be answered in each section of this document. Do not remove any of the text or information provided unless explicitly instructed to do so.

• The total word count for the answers to the assigned questions is intended to be 800 words. This will not be strictly enforced as long as it does not exceed 1000 words in total. Approximate word counts included in square brackets next to the questions have been indicated as a guide only.

Section 1: Evaluating the consequences of mutations on RepX 

The repressor X protein contains multiple regions that are important for its function. Each of these regions is shown on the annotated DNA sequence provided in the Section 1 data. 

• The C6 zinc cluster domain which is required for zinc-binding and binding to DNA is shown in yellow underlined text. The characteristic sequence of the domain is represented as CysX2CysX6CysX5-12CysX2CysX6-8Cys (where X may denote any amino acid). The specific C residues involved in the coordination of the zinc atoms are highlighted in green.

• A linked region adjacent to the zinc finger that is required for recognizing specific target DNA sequences is shown in underlined red text.

• A dimerization domain, through which Rep X forms both homo- and heterodimers that enable DNA binding, is indicated in underlined grey text.

• A C-terminal region of the protein that allows it to function as an activator for some target genes (but not for Enzo) is highlighted in yellow. The acidic and hydrophobic residues are particularly important for this activity.

Section 2: Annotation of the rep gene from S. hypothetical

• Two research students studying the organism S. hypothetical have obtained a DNA sequence for a gene that is believed to be related to repX from S. pretends (gene analyzed in Section 1). 
• The two research students are having a disagreement on the correct annotation of the repX gene. The different DNA sequence annotations (annotations 1 and 2) are shown in the Section 2 data. 
• Both forward and reverse DNA strands are shown, but since the students agree that the coding sequence of the gene is on the top (forward) strand, only three reading frames are shown. 
• The predicted mRNA and protein for Annotation 1 are in purple text, whereas for Annotation 2 they are in orange text.

Section 3: Interaction between genes regulating the expression of the gene for Enzyme P

As mentioned in the background information, Enzyme P is required for the production of compound C, and is repressed by Repressor X. Several other factors are also believed to regulate Enzyme P expression, including repressors Y and Z. Null mutants in the genes coding for Enzyme P and Factors X-Z have been created and assayed for the amount of compound C that is produced (Table 3.1).

To analyze the possible genetic interactions of the factors regulating Enzyme P, pair-wise crosses of the null mutants in repressors X, Y and Z were performed. As mention previously, the lifecycle of S. pretends is similar to that of the baker’s yeast S. cerevisiae. This includes a sexual life-cycle where single-celled haploid parent cells fuse to create a transient diploid, which then undergoes meiosis to produce haploid spores.

The amount of compound C was measured for the progeny of each cross. Progeny were classified into the following phenotypic classes based on the amount in units of Compound C produced (0-0.25; 0.25-0.5; 0.5-0.75; 0.75-1.0; greater than 1.0). The numbers of progeny in each class for each cross is shown in the Section 3 data. 

Q3.1 a: Which of the proposed models (Figure 3.1, Section 3 data) best illustrates how the three repressors are contributing to the regulation of Enzyme P? 

Q3.1 b: Drawing on the data, provide a written justification for your chosen model. If there is more than one plausible model, then explain why this is the case. [Approximately 100-150 words]

Q3.2: What are the linkage relationships between the three genes encoding repressors X, Y and Z? Indicate which of the genes may be linked and provide a brief written explanation for how you have reached this conclusion.

section 4: Investigating gene regulation controlling Compound C production

The research lab is trying to find ways to minimize the amount of compound C that is produced. They have previously determined that the presence of by-product B leads to the dissociation of Repressor X from the enzP promoter. This leads to the derepression of Enzo and the production of more toxic Compound C. They propose to activate the alternate pathway where by-product B is instead broken down to the harmless Compound D. This ‘shunting’ of by-product B into the alternate pathway will reduce the amount that can be converted to Compound C. 

However, they have found that the enzyme responsible for producing Compound D, Enzyme W, is expressed only under very specific conditions. The expression of the Enzo gene requires a transcriptional activator ActQ that is only active as a heterodimer with Repressor X when by-product B is available but is rapidly inactivate once Compound D begins to accumulate. This feedback loop contains the amount of Enzyme W that can be produced. The proposed model for how Repressor X and Activator Q control the expression of the enzymes that produce Compound C and D is proposed in Figure 4.1 (Section 4 data).

The researchers want to investigate whether this model is accurate and how they can create mutants that will lead to increased Compound D and reduced amounts of toxic Compound C. Two mutants have been created. The first (i) is a null mutant in the gene coding for the ActQ protein and the second (ii) is a mutation in the RepX DNA binding site located upstream (in the promoter) of the gene.

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