Gene Technology Exam
Before answering review:
Before answering this part read the History section.
Between 1980-1985 in Sitania (a made-up continent on earth) the entire human population died from TIDS infection, except for a small tribe. A scientific team from the USA went to investigate why this tribe survived TIDS and what could be done to save human beings from a similar future disaster.
The research team consisted of biochemists, molecular biologists , bioinformaticist, geneticists and epidemiologists. For the sake of the exam, several cell lines were isolated from various tissues (donated by one of the tribe elders). It was found that stem cells derived from bone marrow survived when challenged (infected) with HIV, unlike bone marrow derived from a control subject.
In 1989, the biochemist on the team purified proteins from the surviving cells (challenged with TIV) and found out an enzyme preparation that was able to specifically degrade the TIV RNA:DNA hybrid. The team concluded that this enzyme activity prevented AIDS simply because the enzyme digested the DNA-RNA hybrid, thus destroying the HIV pathogen. Following different chromatography steps, the biochemist purified this activity, and noticed three protein bands on SDS-PAGE gels, with molecular masses of 10, 30 and 50 kDa respectively. Upon sequencing of 30 kDa polypeptide, the bioinformatic biologist noticed that the amino-acid sequence derived from the 30 kDa protein had high homology to a well-known NUCLEASE that was isolated and discovered in potato. The enzyme activity of the recombinant NUCLEASE from potato also degrades RNA-DNA hybrids when various viruses infect the potato plant. Everybody in the team said ‘aha’.
A cDNA library isolated from resistant bone marrow cells following exposure to TIV was used to identify the tribe-specific nuclease gene. Following successful identification of the nuclease gene, its corresponding genomic sequence derived from member of the tribe was published in 1990. No other information was provided for the 10 and 50 kDa proteins.
With this great discovery on the TIV-resistant NUCLEASE, the major question was how can we use this information to protect the remaining human race on earth from AIDS.
8 Questions for Part 1:
1 (8 points). The year is 2033, TIBS IS SPREADING. A student who took GeneTech PBIO4500/6500 compared the nucleotide sequence encoding the Tribe- nuclease gene unit with the human genome. She noticed that ALL humans on earth, except the members of the resistant tribe, have identical nuclease alleles (i.e. they are essentially monomorphic). She noticed however, that the sequence of tribe nuclease gene had a nucleotide substitution located in an exon (nucleotide position 545). As a result of that nucleotide mutation, the encoded human NUCLEASE protein will have amino-acid substitution from AGA to AGC.
a– What did the mutation result in?
b– whom would you considered a mutant, members of this tribe or the rest of human race?
- (10 points) The tribe genomic region encoding the 30 kDa NUCLEASE was cloned into phage lambda. The phage harboring the tribe-nuclease gene unit (i.e. GOI) was used to infect E.coli (DH5-alpha) and as a result, the entire Nuclease gene unit is now integrated into the chromosome of E.coli.
A Student who received the engineered bacterium tried to expressthis nuclease gene, but it was not transcribed.
a– How did the student determine whether transcription occurred or not? Provide minimum two names of methods.
b– Provide a possible explanation why transcription failed;
(IMPORTANTNOTE: Answers related to Translation will not be accepted)
- ( 12 points). Provide an answer and the step wise steps you will be using ONLY for the successful transcription of the Eukaryotic nuclease gene in bacteria. NOTE: You cannot use an expression vector (like pET or others we covered in class) for this question. ANOTER NOTE: you do not need to understand how bacteriophage inserts theGOI .
- (12 points) The student solution provided in 3 worked. After correcting the transcription problem, the bacterium transcribes this gene into mRNA, and translates the mRNA into protein but…not efficiently. After protein sequencing of the recombinant protein, the student found unexpected amino acids (aa).
a- If the protein contains a completely different amino-acid sequence when compared with the native nuclease protein, what could be the reason?
– Provide 2 or more possible explanations.
– Provide a possible solution to correct the problem in E.coli based on your explanation. You cannot use a solution that includes an expression vector for this question
b- If the protein you have just expressed in bacterium contains additional amino acids (at the N-terminal) which were not identified in the mature nuclease protein isolated 20 years ago, what could be the reasons? Provide possible explanations and provide a possible solution to correct the problem in E.coli. You cannot use an expression vector for this question
- (12 points) After correcting the translation problem, the bacterium transcribes this nuclease into mRNA, and translates the mRNA into protein. The protein produced is useless and found to contain smaller protein size than does the protein made by the eukaryotic cell.
a– Provide TWO possible explanations (assume no mutation occurred) why the polypeptide is shorter?
b– What methods will you use to correct the problems you have suggested in 5A in E.coli?
c- If the human gene is a mitochondrial gene. Would your answer be any different?
- (8 points) Now a full protein is being made, however at very low amounts. Provide explanations and ways to improve production of the recombinant protein in bacteria.
- (18 points) Presumably, the solution you provided in 6 worked. The protein is expressed at high amounts and it is time to sell the product, and save the world. But before you start counting your dollars you need to purify the protein and of course you need to verify that the protein can degrade the HIV nucleic-acids.
a– How will you purify the NUCLEASE?
b– You test the activity of the pure recombinant NUCLEASE. The protein is not active. Provide several explanations?
- (20 points) You review the literature, and you notice that the biochemist who worked on this problem 20 years ago, isolated three proteins, one of which was the NUCLEASE. You suspect that the other twoproteins (i.e. the 10 and 50 kDA products) may be important for the NUCLEASE activity. Remember other than the size (10, 50 kDa) you have no other information. (i.e. you do not know the 10 and 50 gene or amino-acid sequences),
a– Design an experiment to re-isolate these UNKNOWN 10 and 50 kDa proteins in order to gain the activity of the nuclease protein you have isolated.
(try DRAWING (take picture and attached along with TEXT) [(hint- affi chr.. two-hyb, others..?)
b– If the 10, 50, and 30 kDa are all required for anti-HIV activity how will you prove it? The information obtained from 8a. can be useful here (other methods can be used as well).
3 Questions + bonus for Part 2:
Read carefully the case presented below.
Single Nucleotide Polymorphisms (SNPs) are single nucleotide variations between members of the same species.
Question11 (40 points)
NOTES to answer question 11:
– analyze separately what the consequence of SNP at each of the above seven locations are. You may assume that the ORF is a 999 bp long gene that encodes a sugar-kinase protein with molecular mass of 33,000 Da.
– The assumption is that per gene both alleles are equally transcribed.
For each of the above seven possibilities, answer: (Limit your answer to each case within 1/2 page).
a- If the SNP is a heterozygous SNP, how would the variation in this DNA region affect the amount, length, and properties of RNA and protein? In your answer explain what will happen when SNP is at the UAS, promoter, 5’UTR, exon (IN THE THIRD nucleotide of the CODON), intron, poly A, or at 3’UTR. Answer seven possibilities.
—SHOW a drawing of the predicted size RNA (based on northern blot or RT-PCR);
and Showprotein (based on western blot using antibody raised against the entire protein) could be helpful. (17 points)
b- If the SNP is a homozygous SNP, how would the variation in this region affect the amount and properties of RNA and protein? Explain. (17points)
c– Considering that the SNP variation is present on the FIRST codon in the exon region:
-How would this variation affect the amount and properties of RNA and protein if the SNP is a heterozygous SNP?
-How would this variation affect the amount and properties of RNA and protein if the SNP is a homozygous SNP? Explain. (6 points)
Question 12 (50 points)
Assume some homozygous patients survive but very miserably, with a SNP located in the exon region. This is a new disease never reported or studied. Limit your answer to Question 12 within 2~3 pages.
a- Help the doctor decide which 3 gene technologyexperimental methods he/she will use in order to identify the location of the problem in the body ? (e.g. liver, blood). Describe step by step each experimental method. (12points).
b- The patient you have analyzed took Gene Technology course, and she is not convinced by your findings. Explain how you would validate the data ? (proposed in 12a experiments) and confirm your diagnosis. (10 points)
c- You would like to help the suffering patients. You receive a 100 million dollar government grant to medically treat these patients. Provide 3 rational gene technology methods (only those we covered more recently in class ? (look at the stem cell/animal cloning methodologies) which you can use to help. (12points). Hint you can think on both protein and genes.
d-If the SNP is in the promoter region how you would change your methods in helping patients? (6points).
Question 13. (10 points)
Related to proteomics
Assume the SNP in homozygous individual alters specific amino acids (aa) of the coded enzyme. For each sub-question answer what you predict could happen to the enzyme function if the SNP alters the specific aa represented in cases (a-d). In addition answer for (a-d), how would you examine experimentally your prediction and determine if such modifications are the cause for the patient problems?
Hint: think about properties of AA and modification we discuss in class
a- Ser was modified to Ala . hint. Phos…. And glycosyl ion…
b- Lys was modified to Ala
c- Asparagine, or Threonine were modified
d- The aa for signal-peptide cleavage site was modified
Question 14. (10 points) BONUS
a- what types of protein post-translation modification are considered not safe when producing plant-GMO line?
b- How would you know that such post-translation occurred? (hint ..mass s…….)