I know some of these might be confusing but your help will really help me learn.
ID: 136179 • Letter: I
Question
I know some of these might be confusing but your help will really help me learn. The teacher requires very very detailed answers and he gives nearly no (and I mean literally nearly zero) partial credit so I'm dying here. Thank you sooooooo much, you have no idea.
Also, I am nearly leaglly blind and I am dyslexic so if you could please write clear it would help so much. I just want to learn like everyone else. SUPER SUPER THANK YOU.
QUESTION 3 (10 PTS) You work at a biotechnology company that is trying to cure sickle cell anemia. The first step in your research is to take the normal version of the hemoglobin gene and insert it into E. coli. The goal is to get the bacteria to make normal hemoglobin protein for you to use in your experiments. 3A. You attempt this by amplifying the hemoglobin gene from human DNA and inserting into E coli. Your PCR primers amplify the entire gene, including the promoter, ORF, and terminator. You insert this sequence into the E coli genome, but you can't detect any hemoglobin mRNA. Why? Describe a specific change you could make to the gene sequence you amplified that might fix this problem and then explain why this would work. (3pt) 3B. You have a similar problem getting translation to occur, but eventually are able to alter the hemoglobin gene enough to get the E. coli to make the hemoglobin protein. When you purify the protein from the bacteria, however, you find that it is longer than expected. When you look at the order of amino acids, the first 80 amino acids match the expected sequence but then the sequence drastically diverges from the known hemoglobin amino acid sequence. How can you explain this? (2pt) 3C. You are able to fix this problem as well and finally purify hemoglobin protein from the bacteria. But the hemoglobin protein is too short! You go back and analyze your original PCR reaction and find that 1 out of every 4 products in your reaction has the same C>A mutation How could this mutation yield a shortened protein? Use specific nucleotide sequences in your explanation. (2pts) 3D. You guess that this mutation arose from a polymerase error during the PCR reaction. Based on the fact that 1 of 4 products has the mutation, you can say with good certainty which round of amplification this mutation occurred in. How do you know when the mutation arose? It's probably easiest to draw a cartoon. (3pts)Explanation / Answer
Hemoglobin gene from human DNA and insert into E.Coli
The single base substitution A-T (?6Glu?Val) in the first exon of the ?-globin gene is the defining mutations of the sickle allele. Individuals homozygous for the mutation have the classical sickle cell disease (SCD) genotypes.
The amino acid substitution in ?S-globin allows formation of defective hemoglobin tetramers that polymerize upon deoxygenation.1 Hemoglobin polymerization causes affected red blood cells (RBCs) to lose normal deformability and adopt the archetypal sickle shape. Rigid sickle RBCs prematurely breakdown, damage endothelia, and occlude vasculature, leading to a cascade of hemolysis, ischemia, inflammation, and endothelial injury.
2.-Patients are afflicted with intensely painful episodes, susceptibility to infection, end-organ injury, and early mortality among other sequelae.
3.-Despite its status as a “monogenic” disease, SCD is surprisingly variable in its clinical severity.
4.-At present, the only curative treatment of SCD is allogeneic hematopoietic stem cell (HSC) transplantation. 6-year disease-free survival of >90% has been reported for transplants from HLA-matched sibling donors.5 However, in the United States, <14% of patients have a matched sibling donor.
5.-Transplants with matched unrelated donors are limited by donor availability and immunologic barriers, such as graft rejection and graft-versus-host disease.3 Attempts to extend allogeneic transplant for SCD to alternative donor sources is an area of ongoing effort.
6.-The SCD community has been cautious to embrace allogeneic HSC transplant in part given its short-term morbidity and mortality risks, though nonmyeloablative preparative regimens may help mitigate these risks.
The clinical report is largely reliant on supportive care and hydroxyurea,12 the development of definitive therapies based on genetic manipulation of autologous HSCs would constitute a major advance.
Gene therapy has long been proposed as a potential cure for SCD.
Gene addition strategies
Two main objectives in Gene addition strategies are
1.-safe and efficient gene transfer or correction of long-term repopulating HSCs
2.-high-level, appropriately regulated, stable gene expression. With current progress at the bench and in the clinic, these goals now appear within reach.
The long path to the clinic for SCD gene therapies has been paved by landmark discoveries that have provided important insights into the developmental regulation of the ?-globin gene cluster.
EXPLAINATION OF MUTATION
The alteration of a single nucleotide in the gene for the beta chain of the hemoglobin protein is all it takes to turn a normal hemoglobin gene into a sickle-cell hemoglobin gene.
Single
nucleotide change alters only one amino acid in the protein chain, but the results are devastating.
Beta hemoglobin (beta globin) is a single chain of 147 amino acids.
In sickle-cell anemia, the gene for beta globin is mutated.
The resulting protein still consists of 147 amino acids, but because of the single-base mutation, the sixth amino acid in the chain is valine, rather than glutamic acid. This substitution is depicted in Table 1.
Table 1: Single-Base Mutation Associated with Sickle anemia
Sequence for Wild-Type Hemoglobin
ATG
GTG
CAC
CTG
ACT
CCT
GAG
GAG
AAG
TCT
GCC
GTT
ACT
Start
Val
His
Leu
Thr
Pro
Glu
Glu
Lys
Ser
Ala
Val
Thr
Sequence for Mutant (Sickle-Cell) Hemoglobin
ATG
GTG
CAC
CTG
ACT
CCT
GTG
GAG
AAG
TCT
GCC
GTT
ACT
Start
Val
His
Leu
Thr
Pro
Val
Glu
Lys
Ser
Ala
Val
Thr
Molecules of sickle-cell hemoglobin stick to one another and forms stiicky rods.
These rods cause a person's red blood cells to take on a deformed, sickle-like shape.
They also tend to clog capillaries, causing an affected person's blood supply to be cut off to various tissues,including the brain and the heart.
When an afflicted individual exerts himself or herself even slightly, he or she often experiences terrible pain, and he or she might even undergo heart attack or a stroke.
RELATION BETWEEN MUTATION AND POLYMORPHISM
Single nucleotide polymorphism (SNP) to refer to a single base pair alteration that is common in the population.
A polymorphism is any genetic location at which at least two different sequences are found, with each sequence present in at least 1% of the population.
The term "polymorphism" is generally used to refer to a normal variation, or one that does not directly cause disease, the cutoff of at least 1% prevalence for a variation to be classified as a polymorphism is somewhat arbitrary, f the frequency is lower than this, the allele is typically called as mutation.
They are important as markers, or signposts, for scientists to use when they look at populations of organisms in an attempt to find genetic changes that predispose individuals to certain traits, including disease.
Sequence for Wild-Type Hemoglobin
ATG
GTG
CAC
CTG
ACT
CCT
GAG
GAG
AAG
TCT
GCC
GTT
ACT
Start
Val
His
Leu
Thr
Pro
Glu
Glu
Lys
Ser
Ala
Val
Thr
Sequence for Mutant (Sickle-Cell) Hemoglobin
ATG
GTG
CAC
CTG
ACT
CCT
GTG
GAG
AAG
TCT
GCC
GTT
ACT
Start
Val
His
Leu
Thr
Pro
Val
Glu
Lys
Ser
Ala
Val
Thr