Athena Williams was born as a healthy baby girl- and while she was small (25th percentile) the family pediatrician wasn’t worried as both her parents were petite. However at about the age of 13 months Athena stopped running around, tired easily, was pale and lost her appetite- symptoms that could not be explained by a recent flu bug going around as it lasted for months. Concerned the family pediatrician Dr. Wright decided to draw Athena’s blood to check for inherited blood-disorders. At the next office visit, Dr. Wright sat Athena’s mother down and gently gave her the bad news. As suspected it was determined that Athena had Thalassemia- an inherited disorder resulting from a defective for β-globin protein- and thus defective Hemoglobin [Like sickle cell anemia]. β-thalassemia patients are treated with frequent transfusions requiring many hospital visits, but this is not a cure. Individuals with severe forms of β-thalassemia often die in mid-teens. Dr. Wright suggested to Athena’s parents that she may be a candidate for experimental gene therapy but first they needed a quick test to see if Athena had the kind of defect that would qualify for gene therapy. They isolated mRNA from Athena’s RBCs and from a person without Thalassemia (N) and performed hybridization experiments. They allowed Athena’s mRNA to hybridize with a clone carrying the genomic DNA of b-globin gene. They did the same with mRNA from person with normal b- globin gene. Shown on the following page is the cartoon of what was observed by electron microscopy of the RNA-DNA hybrid. Double-stranded regions (hybridized) are represented by thick lines, and single-stranded regions (un-hybridized) are represented by thin lines Dr. Wright needed to check the exact nature of the mutation in Athena’s beta-globin gene to determine if Athena would be a good candidate for gene therapy. Sequencing a region of the Beta-globin gene covering exon 2- intron 2- exon 3 revealed the following mutations within intron 2 when compared to normal beta globin gene (mutations underlined). a) How is Athena’s intron different from the normal β-globin pre-mRNA? (Use terms such as 5′ splice site, 3’ splice site, etc.) b) Assuming that the cellular splicing machinery will recognize sets of “GU” and “CAG” together in the right context as marking the beginning and end of an intron, predict how Athena’s pre-mRNA will be processed into mRNA as a result of the mutation. Do this by drawing what the resulting mRNA sequence from this process will look like. Write out the sequence, box the exon sequences and label 5’ and 3’
Athena Williams was born as a healthy baby girl- and while she was small (25th percentile) the family pediatrician wasn’t worried as both her parents were petite. However at about the age of 13 months Athena stopped running around, tired easily, was pale and lost her appetite- symptoms that could not be explained by a recent flu bug going around as it lasted for months. Concerned the family pediatrician Dr. Wright decided to draw Athena’s blood to check for inherited blood-disorders.
At the next office visit, Dr. Wright sat Athena’s mother down and gently gave her the bad news. As suspected it was determined that Athena had Thalassemia- an inherited disorder resulting from a defective for β-globin protein- and thus defective Hemoglobin [Like sickle cell anemia].
β-thalassemia patients are treated with frequent transfusions requiring many hospital visits, but this is not a cure. Individuals with severe forms of β-thalassemia often die in mid-teens. Dr. Wright suggested to Athena’s parents that she may be a candidate for experimental gene therapy but first they needed a quick test to see if Athena had the kind of defect that would qualify for gene therapy.
They isolated mRNA from Athena’s RBCs and from a person without Thalassemia (N) and performed hybridization experiments. They allowed Athena’s mRNA to hybridize with a clone carrying the genomic DNA of b-globin gene. They did the same with mRNA from person with normal b- globin gene.
Shown on the following page is the cartoon of what was observed by electron microscopy of the RNA-DNA hybrid.
Double-stranded regions (hybridized) are represented by thick lines, and single-stranded regions (un-hybridized) are represented by thin lines
Dr. Wright needed to check the exact nature of the mutation in Athena’s beta-globin gene to determine if Athena would be a good candidate for gene therapy. Sequencing a region of the Beta-globin gene covering exon 2- intron 2- exon 3 revealed the following mutations within intron 2 when compared to normal beta globin gene (mutations underlined).
a) How is Athena’s intron different from the normal β-globin pre-mRNA? (Use terms such as 5′ splice site, 3’ splice site, etc.)
b) Assuming that the cellular splicing machinery will recognize sets of “GU” and “CAG” together in the right context as marking the beginning and end of an intron, predict how Athena’s pre-mRNA will be processed into mRNA as a result of the mutation.
Do this by drawing what the resulting mRNA sequence from this process will look like.
Write out the sequence, box the exon sequences and label 5’ and 3’
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