Download ALE 8 - Biol 100

Answer Key
Biology 100
Instructor: K. Marr
ALE #10. From Gene to Protein and Biotechnology Practice Problems
1. Compare the differences in RNA and DNA by completing the table below.
DNA
RNA
Number of Strands
2
1
Name of sugar in
nucleotides
deoxyribose
Ribose
Bases present
Adenine, thymine,
cytosine, guanine
Adenine, uracil, cytosine, guanine
Where produced in
cell
Nucleus (during DNA
replication)
Nucleus (during transcription)
Each of these kinds of RNA are produced in
the nucleus
mRNA: in the cytoplasm and attached to
ribosomes during translation
Where found in cell
Nucleus
(also in mitochondria and tRNA: cytoplasm—carries amino acids to
the ribosomes during translation.
chloroplasts)
rRNA: a component of ribosomes found on
the rough ER and free floating ribosomes in
the cytoplasm
Name of process that
makes it
2.
DNA replication
Transcription
Fill in the following spaces concerning the ―central dogma‖ of biology: The __coding___ strand of a gene is a
long chain _____nucleotides______. The order of ____nucleotides
controls the order of _____
(or bases)____ in a gene
nucleotides (or bases)____ in mRNA, which determines the order of
___amino
acids_____ in the protein the gene codes for, which controls the
____shape (or structure)___ of the protein, which in turn determines the function of that protein. An
organism’s genotype determines the kind of proteins an organism can make. These proteins determine an organism’s
____phenotype_______.
3.

Outline the flow of genetic information from DNA to the production of proteins: DNA  RNA  Protein. For each
step indicate where it takes place in the cell, the name of each process involved, what is needed for each process to
occur, the names of the major enzymes involved, etc.
DNA  mRNA  Protein
Transcription is the process where DNA acts as a template for the production of RNA; Occurs in the nucleus of
a cell with the help of RNA polymerase and RNA nucleotides. mRNA leaves the nucleus for ribosomes, either
free floating in the cytoplasm or to those on the rough E.R. RNA polymerase is the enzyme responsible for
separating the coding and noncoding strands of the gene to be transcribed and for transcribing the coding
strand matching A, U, G and C RNA nucleotides to the DNA nucleotides T, A, C and G, respectively.
ALE #10 – Revised W2011–Page 1 of 6

Translation (Protein Synthesis). During protein synthesis, ribosomes move along the mRNA molecule and
"read" its sequence three nucleotides at a time (codon) from the 5' end to the 3' end. Each amino acid is
specified by the mRNA's codons. Each codon pairs with a specific anticodon, a sequence of three
complementary nucleotides at one end of a tRNA molecule. Since each tRNA molecule carries a specific amino
acid at one end, the order of codons on the mRNA molecule determines the order of amino acids to be linked
together during protein synthesis.
4.
Here is a hypothetical gene showing the sequence of DNA nucleotides for the coding strand (i.e. the coding strand is
the strand that is transcribed). IMPORTANT!! This sequence includes the regions that code for start and stop in
translation—that is, locate both the start codon and stop codon in the mRNA before you translate it into protein!!
Coding strand of DNA:
3’ T A G G T A C T G G G G C A T T A A 5’
a.
What would be the sequence of nucleotides in the resulting mRNA if this strand of DNA was transcribed? Label
the 5’ and 3’ ends of the mRNA molecule.
mRNA:
5’ AUCCAUGACCCCGUAAUU 3’
Start Codon
b.
What is the amino acid sequence of the protein that this gene codes for? (You will need the table of codons in your
textbook to answer this question.)
The amino acid sequence is:
5.
Stop Codon
Met - Thr - Pro
Hemoglobin is a protein in your red blood cells that is responsible for carrying oxygen. A mutation in the gene that
codes for hemoglobin leads to a disease called sickle cell anemia. Sickle cell hemoglobin is unable to carry oxygen
effectively, resulting in weakness in individuals who inherit one copy of this gene, death in results if the faulty gene
is inherited by from both parents. There are over 300 known mutations in the hemoglobin genes. One of these
mutations causes a condition called Hemoglobin C disease, which is not as serious as sickle cell anemia. You will
need the table of codons in your book to answer some of these questions.)
a. Below is the part of the sequence from the coding strand of DNA for three variants of the hemoglobin gene. Circle
the mutation in the sickle cell sequence and the hemoglobin C sequence.
b.
sickle cell anemia:

hemoglobin C disease: 3’...T G A G G A C

normal hemoglobin:
3’...T G A G G A
What is the mRNA sequence of the normal hemoglobin gene? Indicate 3’ and 5’ ends.
5’… ACUCCUGAGGAG…3’
What is the amino acid sequence of the normal hemoglobin gene?
The amino acid of normal hemoglobin:
d.
A C C T C...5’
3’...T G A G G A C T C C T C...5’
mRNA of normal hemoglobin:
c.
T T C C T C...5’

Thr – Pro – Glu – Glu
Exactly what is the difference between the normal hemoglobin protein and the hemoglobin protein in a person with
sickle cell anemia and a person with hemoglobin C disease?

normal hemoglobin vs. sickle cell:
The 3rd amino acid, Glu, in normal hemoglobin is changed to Lys in sickle cell hemoglobin

normal hemoglobin vs. hemoglobin C:
The 3rd amino acid, Glu, in normal Hemoglobin is changed to Val in Hemoglobin C
ALE #10 – Revised W2011–Page 2 of 6
e.
As mentioned above, there are over 300 known mutations in the hemoglobin genes. While many of these mutations
lead to diseases, some of these mutations do not change the ability of the hemoglobin protein to do its job.
Describe how it is possible that a mutation in the hemoglobin gene doesn't affect how the hemoglobin protein
works.
There are two possibilities:
 Some DNA nucleotide changes result in no change in the protein because there is a
redundancy in the genetic code. E.g. GCA, GCU, GCG, and GCC all code for the
same amino acid, alanine.
 Some nucleotide changes result in a change in an amino acid in some part of a
protein that is not critical to the function of the protein
6.
Suppose you are a physician interested in cloning the CFTR gene from the cheek cells of healthy people to use in gene
therapy trials to treat patients with cystic fibrosis. Starting with a small sample of human DNA (i.e. human cheek cells
on a cotton swab) explain how PCR (Polymerase Chain Reaction) could be used to make millions of copies of the
CFTR gene. Your explanation should include the roles of each of the following:
 primers,
 taq DNA polymerase (a heat-stable DNA polymerase from a bacterium that lives in hot springs),
 DNA nucleotides,
 repeated cycling of temperatures: 94oC to 55oC to 72 oC and back to 94 oC.
 In addition to an explanation, include a labeled diagram that shows three cycles of the PCR process. You’ll
find a discussion and illustration of PCR on page 177 in your textbook, Essentials of Biology by Mader 2nd ed.
Use these pages only as a reference and guide—do not copy this information, rather, reformulate it in your
own words.
The process of cloning the CFTR gene
from the small sample of cheek cells
would involve using detergent to lyse
(break open) the cheek cells to release
their DNA. The sample would then be
heated to near boiling (~94 oC) for a
couple of minutes to separate the doublestranded DNA into single-stranded DNA,
cooling to ~55oC for a couple of minutes
to allow the DNA primers to hydrogen to
regions of the coding and noncoding
strands just outside of the CFTR gene,
then cooling to ~72 oC to allow the heat
stable taq DNA polymerase to copy the
CFTR gene. This process would be
repeated over and over again through 55
or 60 cycles, producing millions of copies
of the CFTR gene.
In the diagram, “targeted sequence,”
refers to the CFTR gene, the gene that
we want to clone, while “primers” refer
to two different DNA primers are
complementary to both the coding and
noncoding strands just outside of the
target sequence, the CFTR gene.
ALE #10 – Revised W2011–Page 3 of 6
7. Good work! You’ve cloned the CFTR gene! Now you wish to use it in gene therapy trials to help your patients. The
following questions pertain to the use of gene therapy in an attempt to cure them of cystic fibrosis. For a discussion
of gene therapy, see Section 13.3 in your textbook, Essentials of Biology by Mader 2nd ed.
a. Describe the steps that would have to occur at the cellular level that would be needed to cure a patient with cystic
fibrosis using gene therapy. Assume the usage of a nasal spray that contains a viral vector containing the normal
CFTR gene.
The nasal spray would contain a vector that contains a normal CFTR gene. A disabled
DNA virus (e.g. adenovirus) or liposome are possible vectors as they insert genes better
in cells that reproduce slowly than do retroviruses (RNA viruses). The vector would have
to enter the lung cells containing the faulty CRTR gene and then insert the normal gene
into one of the chromosomes. The CFTR gene would have to be transcribed and the
resulting mRNA would need to be translated to produce the normal CFTR protein. The
CFTR protein would need to be produced in the correct amounts and at the correct times
in the life cycle of all cells that it is inserted into
b. Would this really be a cure? Explain why or why not.
Yes and no—yes it would be a permanent cure for all cells that have met the criteria
mentioned above—No it wouldn’t be a permanent cure for the cells that do not
contain the healthy CFTR gene or don’t produce the correct amounts of the CFTR
protein or don’t get this protein into the plasma membrane.
c.
How would you use gene therapy to insure that the descendants of a CF patient don’t have to worry about
getting this genetic disease?
Germline gene therapy would be necessary—that is, egg cells would have to have the
normal gene inserted into them by gene therapy. Due to their small size, gene therapy
as yet to be shown effective in the genetic modification of sperm cells.
d.
What are the challenges facing gene therapy that prevent it from working? Explain.
Gene therapy faces many challenges:
 Getting vector to most if not all of the desired cells
 Getting the gene of interest inserted into the one of the chromosomes of the desired
cells without disrupting normal functioning genes in the target cells (could lead to
cancer or other genetic disorders if genes are disrupted)
 Getting the gene of interest to be transcribed and the resulting mRNA translated
in the correct amounts and at the correct times
 Getting the desired gene to remain inserted in the chromosomes of the desired
cells.
e.
What are the potential dangers of gene therapy to the patient? Explain.



Disruption of normal functioning genes due to the random insertion of the gene of
interest could result in cancer if oncogenes are affected or other genetic disorders
Disease due to the possible mutation of the disabled vector (e.g. a retrovirus) into a
virulent (disease causing) form of the vector.
Severe immune reaction to the vector that could lead to death (e.g. Jesse Gelsinger
in Fall of 1999.
ALE #10 – Revised W2011–Page 4 of 6
Connections with Cell Structure and Function….
8. Cystic fibrosis is due to the inheritance of two faulty CFTR genes. The normal CFTR gene codes for a membrane
protein found in cells involved with secretion, for example: mucous secreting cells in the respiratory tract, sweat glands
in the skin, digestive enzyme secreting cells of the pancreas, etc. The CFTR protein is actually a glycoprotein, that is, it
is a protein that has been modified to by the addition of several monosaccharides.
a. Trace the pathway of the normal CFTR protein from the organelle where the CFTR protein is made to its final
location in the cell membrane. Hints: What cell organelle makes proteins? Where is this organelle located,
cytoplasm or rough ER? Where are the sugar molecules added to the CFTR protein? How does the CFTR protein
get there? How does the CFTR get to the plasma membrane?
Rough E.R.  Vesicle  Golgi Apparatus  Vesicle  Plasma membrane
b.
Make a sketch of a cell and trace the pathway taken by the normal CFTR protein from where it is made to the
plasma membrane.
c.
The function CFTR protein is to pump chloride ions (i.e. salt) into the cells lining the lungs and cells lining various
ducts in the body. In cystic fibrosis, the CFTR protein is defective never makes it to the plasma membrane of these
cells. As a consequence salt concentration increases resulting in a hypertonic solution in the alveoli (air sacs) of
the lungs, the ducts that carry sweat from sweat glands to the skin, oviducts, vas deferens, etc. Use this information
to explain why people with CF experience the following symptoms:
 Salty sweat
Since the plasma membranes of the cells lining the ducts of the sweat glands lack the
CFTR protein, these cells can’t pump salt into these cells. This results in the sweat
containing more salt than normal.
 Mucous collects in the lungs
Since the plasma membranes of the cells lining the lungs lack the CFTR protein, these
cells can’t pump salt into the lung cells from the air sacs within the lungs. This results in
salt buildup in the air sacs of the lungs which draws a mucous-rich fluid into the lungs—
just like pouring salt on a slug stimulates the release of a mucous-rich fluid.
 Chronic respiratory infections
Bacteria thrive in the warm, moist, carbohydrate-rich mucous in the lungs—hence
chronic respiratory infections.
 Problems digesting food
Since the plasma membranes of the cells lining the pancreatic duct (the duct that carries
digestive enzymes from the pancreas to the small intestine) lack the CFTR protein, these
cells can’t pump salt into these cells. This results in salt buildup in the pancreatic duct
ALE #10 – Revised W2011–Page 5 of 6
which draws a mucous-rich fluid into the duct. The mucous physically hinders the
transport of digestive enzymes to the digestive tract, hence the difficulty in digesting food.
 Reduced fertility in women
Oviducts (fallopian tubes) are partially clogged with mucous for the same reasons as
indicated above for the pancreatic duct. Hence, the reduced fertility since sperm must
pass through the oviducts to fertilize the egg. Moreover, the fertilized egg must pass
through the oviduct in order to implant in the uterine wall.

The vas deferens (the duct that carries sperm from the testes to the
urethra and then to the outside world) are partially clogged with mucous for the same
reasons as indicated above for the pancreatic duct. Hence, the reduced fertility. Some
men with cystic fibrosis lack the vas deferens entirely—hence no fertility.
Sterility in males
ALE #10 – Revised W2011–Page 6 of 6