Deck 10: The Integration of Genetic Approaches: Understanding Sickle Cell Disease

Question 1

Hemoglobin, the protein responsible for binding and transporting oxygen, forms a tetramer. What is the protein composition of the most common form of hemoglobin, hemoglobin A (HbA)?

A)α₁β₁
B)α₁β₂
C)α₂β₁
D)α₂β₂
E)α₂β^S

Answer 1

D

Question 2

Molecular charge is an important characteristic influencing protein migration on a gel. What accounts for the variation in the molecular charge of proteins?

A)the intronic sequences of the protein
B)the amino acid composition of the protein
C)the negative charge of the sugar-phosphate backbone
D)the amount of protein loaded into the wells of the gel
E)the presence of secondary structures in the protein

Answer 2

B

Question 3

The mutation that causes SCD causes what kind of change in the structure of hemoglobin?

A)decreases heme-binding ability
B)fewer chains of amino acids (trimeric rather than tetrameric structure)
C)a single amino acid substitution that alters the structure of the tetramer
D)addition of a single amino acid that causes a longer protein subunit
E)loss of a single amino acid that causes a frameshift and premature stop codon

Answer 3

C

Question 4

How is the DNA visualized on a Southern blot?

A)ethidium bromide
B)radioactively labeled probes
C)chemically labeled probes
D)both ethidium bromide and chemically labeled probes
E)either radioactively labeled probes or chemically labeled probes

Question 5

As part of the maturation process, red blood cells lose their nucleus. What is the consequence for cell division in these cells?

Question 6

Two probes, A and B, hybridize very near each other in a region of DNA that contains RFLPs when digested with HindIII. The four maps show the location and intervening distances of the HindIII restriction sites and the binding locations of probes A and B. The maps show alleles H¹-H⁴ present in a population. What band(s)is/are seen in Southern blot analysis of a H²H³ heterozygote using probe B?

A)3 kb and 5 kb
B)3 kb and 8 kb
C)3kb, 2kb, and 8 kb
D)5 kb and 8 kb
E)2 kb, 3 kb, and 5 kb

Question 7

You set up an EcoRI restriction digest for a sample of genomic DNA from an individual with a mutation in the β-globin gene. You perform a Southern blot using the entire β-globin gene as a probe. To your surprise, you notice a different pattern of bands in the affected individual - you have identified a new SNP! What can you conclude from the pattern of bands from the Southern blot below?

A)The SNP resulted in the loss of an EcoRI site.
B)The SNP resulted in the creation of a new EcoRI site.
C)The SNP resulted in the loss of an exon.
D)The SNP results in the creation of a new HindIII site.
E)There was no change in the genomic DNA sequence.

Question 8

Sickle cell is a codominant disease, meaning that heterozygotes exhibit an intermediate blood cell phenotype (a mixture of normal and sickle cells)due to the blend of hemoglobin tetramers in each red blood cell. Which of the following tetramer structures would you expect to find in a carrier of SCD?

A)α₂β^A₂
B)α₂β^Sβ^A
C)α₂β^S₂
D)α₂β^A₂ and α₂β^Sβ^A
E)α₂β^A₂ and α₂β^Sβ^A and α₂β^S₂

Question 9

Which of the following techniques describes either the rate of a molecule's migration or its final position in a gel?

A)hybridization
B)electrophoretic mobility
C)densitometry
D)restriction fragment analysis
E)peptide fingerprint analysis

Question 10

Given what you know of the inheritance pattern of SCD, what can be said of individual II-5?

A)He is a carrier because his mother was affected and his son was affected.
B)His children with II-4 will have a 50% chance of being carriers (β^Aβ^S).
C)His children with II-4 will all have a 50% chance of being affected (β^Sβ^S).
D)A and B are correct.
E)A, B, and C are correct.

Question 11

Which of the following techniques quantifies the amount of protein present in a gel lane by measuring how much light is blocked from passing through the gel by the presence of a band of protein?

A)hybridization
B)electrophoretic mobility
C)densitometry
D)restriction fragment analysis
E)peptide fingerprint analysis

Question 12

Given what you know of the inheritance pattern of SCD, is the following pedigree representative of a typical SCD pedigree?

A)Yes; this is a pedigree showing dominant inheritance, and SCD exhibits dominant inheritance.
B)Yes; this is a pedigree showing recessive inheritance, and SCD exhibits recessive inheritance.
C)No; this is a pedigree showing dominant inheritance, and SCD exhibits recessive inheritance.
D)No; this is a pedigree showing recessive inheritance, and SCD exhibits dominant inheritance.
E)Another generation is needed before you can determine mode of inheritance.

Question 13

Can Southern and northern blotting detect differences between WT and mutant β-globin gene or between WT and mRNA or both?

A)Only differences between WT and the gene can be detected.
B)Only differences between WT and the mRNA can be detected.
C)Differences between the WT and both the gene and the mRNA can be detected.
D)Neither differences between WT and the gene nor between WT and the mRNA can be detected.

Question 14

You are interested in designing a molecular probe to detect the region of β^S SNP, which includes a DdeI (CTNAG)restriction site. Which of the following molecular probe sequences would anneal to the correct position?

A)ACTCCTGAGGAGAAG
B)TGAGGACTCCTCTTC
C)GTGCACCTGACTCCT
D)CACGTGGACTGAGGA
E)CACGUGGACUGAGGA

Question 15

At a biological level, β^Aβ^S heterozygotes suffer fewer cases of malaria than β^Aβ^A homozygotes, and when they do get malaria, their disease is less severe. What is the biological explanation for the heterozygous advantage?

A)The β^Aβ^A homozygotes have a shortened average life span of their red blood cells, and the shorter RBC life spans interrupt the developmental cycle of Plasmodium larvae by causing the parasites to mature and reproduce rapidly.
B)The β^Aβ^S heterozygotes have a shortened average life span of their red blood cells, and the shorter RBC life spans interrupt the developmental cycle of Plasmodium larvae by preventing many of the immature parasites from reaching maturity.
C)The sickle-cell shape of the RBCs in β^Aβ^S heterozygotes causes the Plasmodium larvae to become stuck in the RBC aggregates, which prevents reproduction.
D)The anemia associated with SCD makes the β^Aβ^S heterozygotes less attractive to mosquitoes, so they experience fewer mosquito bites and less opportunity for infection by Plasmodium larvae.
E)The β^Aβ^S heterozygotes are bitten by mosquitoes less frequently, and when they are infected, the sickle-cell shape of the RBCs in β^Aβ^S heterozygotes causes the Plasmodium larvae to become stuck in the RBC aggregates, which prevents reproduction.

Question 16

From a standpoint of natural selection, we see that the heterozygous advantage for β^Aβ^S individuals is balanced by the disadvantage to those with SCD or the wild-type alleles (and no malaria protection). The action of these conflicting trends produces an overall increase in the mutant allele until it reaches a state where the gain and loss of β^S alleles is equal. What is the term for this stable condition that allows mutant alleles to remain in a particular population?

A)equilibrium frequency
B)heterozygote fitness
C)restriction fragment length polymorphism
D)single nucleotide polymorphism
E)balanced polymorphism

Question 17

What term is used to describe the situation when the loss of an allele because of selection against one of its phenotypes is balanced by natural selection in favor of the allele for another phenotype?

A)equilibrium frequency
B)heterozygote fitness
C)restriction fragment length polymorphism
D)single nucleotide polymorphism
E)balanced polymorphism

Question 18

A couple has four children; one is affected by SCD, and two are carriers of the disease. What can be said of the genotypes of the parents?

Question 19

Thalassemia is a recessive blood disease caused by reduced synthesis of one of the globin genes. While SCD is a mutation in the structure of hemoglobin, thalassemias can also be caused by mutations in the regulatory elements, causing too little globin to be produced and resulting in anemia. You've designed a DNA probe that you want to use to screen a family for a rare mutation in the β-globin gene that causes thalassemia. First you will use the probe for northern blotting. What information will the northern blot tell you?

A)whether the mRNA for β-globin is being translated into functional proteins in a given cell population
B)whether the amount of the β-globin gene being transcribed (mRNA levels)is within normal range
C)the size of the mRNA being transcribed
D)the presence of a particular mutation by restriction fragment analysis
E)both B and C are correct

Question 20

Ingram examined hemoglobin structural variation by breaking down the hemoglobin protein into many fragments by chemical treatment and subjecting the fragments to electrophoresis to separate them in a gel; and then in a second dimension, perpendicular to the first, he separated them by chromatography. This two-step approach is commonly referred to as __________.

A)hybridization
B)electrophoretic mobility
C)densitometry
D)restriction fragment analysis
E)peptide fingerprint analysis

Question 21

You have digested a molecule of DNA and want to identify a specific fragment of interest. The DNA is subjected to gel electrophoresis, but you get two bands that are very close in size. What could you use to determine which band is the correct one?

Question 22

Which of the following images represents the expected results of a DNA gel for three individuals with different genotypes-homozygous dominant (lane 1), heterozygote (lane 2), and homozygous recessive (lane 3)?

Question 23

A restriction map of β^A and β^S is shown. Members of a family were analyzed by cutting their DNA with DdeI. Which individuals in the family have the genotype β^Aβ^A?

Question 24

Given the following plasmid, what are the sizes of the fragments you would expect from a NotI digest?

Question 25

The rate of molecule movement in electrophoretic gels depends on three parameters of molecular structure. Each of these parameters individually is important in determining how a particular molecule migrates, but they can also interact with one another to produce a characteristic migration rate for each molecule. What are the three parameters?

Question 26

The bone marrow must continuously generate new red blood cells because the life span of a typical red blood cell is only ________. The life span is even shorter in individuals with sickle-cell trait, which accounts for the protective effects against malaria.

Question 27

Based only on this Southern blot and with no knowledge of which of the β-globin SNPs you are looking at, which lane(s)might represent(s)a heterozygous individual?

Question 28

Given the following plasmid, what are the sizes of the fragments you would expect from an XhoI digest?

Question 29

Given the following gel, which DNA fragment has a lower electrophoretic mobility?

Question 30

Given the following gel, which DNA fragment has a lower molecular mass?

Question 31

Hemoglobin is made up of four proteins from two different gene clusters, the α-globin gene cluster and the β-globin gene cluster. The two clusters are related, having evolved from a common ancestral gene through gene duplication, based on strong similarities in which three gene characteristics?

Question 32

Given the following gel, which protein has a more negative charge?

Question 33

Sodium dodecyl sulfate (SDS)is a detergent commonly added to protein samples before gel electrophoresis. The purpose of the negatively charged SDS is to neutralize the charges and denature the protein to remove secondary structures. Thus, the proteins can be separated on a gel based solely on what parameter?

Question 34

Before transferring DNA from a gel to the membrane in Southern blotting, the DNA must be denatured (usually by soaking the gel in NaOH). Why is this step necessary?

Question 35

Globin genes contain ________ segments that ultimately encode the amino acids of the protein and interspersed ________ sequences that do not contribute to encoding the protein product of the gene.

Question 36

A SNP results in a loss of the XhoI site indicated in the bacterial plasmid below. You design a molecular probe that spans the lost XhoI site. Which lane represents the expected Southern blot of a bacterial cell that carries two copies of the plasmid: a normal version and the version with the lost XhoI site?

Question 37

Given the following Southern blot results, which lane represents a heterozygous individual?

Question 38

The different globin genes in each cluster are sequentially expressed in tightly controlled patterns during the various stages of prenatal and adult development. What gene structure helps regulate globin gene transcription by controlling both the timing and the starting point for transcribing DNA into mRNA?

Question 39

What information allows you to predict the pattern of DNA bands expected in a Southern blot of an individual with the wild-type allele?

Question 40

Given the following gel, which protein is more likely to have a globular shape?

Question 41

The intercalating agent ________ allows researchers to visualize DNA and RNA on a gel when the gel is exposed to UV light.

Question 42

Burkitt's lymphoma is a cancer of the lymphatic system. The endemic variant, common to equatorial Africa, commonly affects children who are also affected by chronic malaria. A recent study found that individuals with AA hemoglobin were twice as likely to develop Burkitt's lymphoma as those with the AS genotype. We saw a similar benefit of certain genotypes in cases
of sickle cell disease. Recall that individuals who are β^Aβ^S, β^Aβ^C, or β^Aβ^E have a survival and reproductive advantage over individuals who are homozygous β^Aβ^A (and therefore succumb more easily to malaria)and over those who are homozygous for the mutant alleles (and therefore suffer from hereditary anemia). Resistance to malaria and Burkitt's lymphoma are both an example of ________ advantage.

Question 43

Northern blotting is not useful in detecting the SNPs associated with the β^A and β^S alleles. Explain why.

Question 44

Being a carrier of the sickle cell trait, although generally not a serious problem, can pose significant risks for athletes, particularly those in intense training regimens. The death of Florida State University linebacker Devaughn Darling, a carrier of the sickle cell trait, in a spring conditioning drill refocused attention on sickle cell abnormalities, including both sickle cell disease and sickle cell trait (carriers). High altitude, aggressive exercise, and high temperatures can all lead to acute rhabdomyolysis in individuals with sickle cell trait. Why do you think an individual who is a carrier of the sickle cell trait may be more likely to experience the symptoms of SCD during rigorous exercise?

Question 45

We saw that Southern blotting of DNA from the β^A allele produces two DNA bands corresponding to fragment lengths of 1150 bp and 200 bp based on the location of two DdeI sites. In contrast, Southern blot analysis of β^S-allele DNA produces a single DNA restriction fragment, measuring 1350 bp in length, since one of the DdeI sites is altered by SNP variation. Why is Southern blotting necessary to identify the correct bands-if you know the sizes, why can't you just identify your band on a gel? Why is the location of the probe (spanning the predicted lost DdeI site)important?

Question 46

Describe the hypothesis that James Neel proposed to describe the inheritance pattern of SCD. What experimental methods did he use to prove his hypothesis?

Question 47

We can detect the SNP in the β^S allele because it destroys a DdeI restriction site, leading to DNA restriction fragment variation revealed by Southern blot analysis.
Either two or three restriction sequences for the restriction endonuclease DdeI can occur near the
β-globin gene, depending on the allele. One DdeI restriction sequence is eliminated from the β^S allele by the SNP in the mutant allele because of a mutation in the double-stranded target sequence 5′ - CTNAG - 3′.
Suppose the wild-type allele sequence is CCTGAG, while a newly identified SNP is CCTTAG. Would this same restriction fragment analysis technique be appropriate for detecting this particular SNP? What other technique(s)could you use to identify this particular mutation?

Question 48

In contrast to the nucleic acid probes used to detect DNA or RNA target sequences, molecular probes used to detect target proteins in western blots are ________.