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Biology - Genetics The Cystic Fibrosis Gene
Introduction:
Cystic fibrosis is an inherited autosomal recessive disease that exerts its main effects on the digestive system and the lungs. This disease is the most common genetic disorder amongst Caucasians. Cystic fibrosis affects about one in 2,500 people, with one in twenty five being a heterozygote. With the use of antibiotics, the life span of a person afflicted with CF can be extended up to thirty years however, most die before the age of thirteen.1 Since so many people are affected by this disease, it's no wonder that CF was the first human genetic disease to be cloned by geneticists. In this paper, I will be focusing on how the cystic fibrosis gene was discovered while at the same time, discussing the protein defect in the CF gene, the bio-chemical defect associated with CF, and possible treatments of the disease. Finding the Cystic Fibrosis Gene: The classical genetic approach to finding the gene that is responsible for causing a genetic disease has been to first characterize the bio-chemical defect within the gene, then to identify the mutated protein in the gene of interest, and finally to locate the actual gene. However, this classical approach proved to be impractical when searching for the CF gene. To find the gene responsible for CF, the principle of reverse genetics was applied. Scientists accomplished this by linking the disease to a specific chromosome. After this linkage, they isolated the gene of interest on the chromosome and then tested its product.2 Before the disease could be linked to a specific chromosome, a marker needed to be found that would always travel with the disease. This marker is known as a Restriction Fragment Length Polymorphism or RFLP for short. RFLP's are varying base sequences of DNA in different individuals which are known to travel with genetic disorders.3 The RFLP for cystic fibrosis was discovered through the techniques of Somatic Cell Hybridization and through Southern Blot Electrophoresis (gel separation of DNA). By using these techniques, three RFLP's were discovered for CF; Doc RI, J3.11, and Met. Utilizing in situ hybridization, scientists discovered the CF gene to be located on the long arm of chromosome number seven. Soon after identifying these markers, another marker was discovered that segregated more frequently with CF than the other markers. This meant the new marker was closer to the CF gene. At this time, two scientists named Lap-Chu Tsui and Francis Collins were able to isolate probes from the CF interval. They were now able to utilize to powerful technique of chromosome jumping to speed up the time required to isolate the CF gene much faster than if they were to use conventional genetic techniques.3 In order to determine the exact location of the CF gene, probes were taken from the nucleotide sequence obtained from chromosome jumping. To get these probes, DNA from a horse, a cow, a chicken, and a mouse were separated using Southern Blot electrophoresis. Four probes were found to bind to all of the vertebrate's DNA. This meant that the base pairs within the probes discovered contained important information, possibly even the gene. Two of the four probes were ruled out as possibilities because they did not contain open reading frames which are segments of DNA that produce the mRNA responsible for genes. The Northern Blot electrophoresis technique was then used to distinguish between the two probes still remaining in order to find out which one actually contained the CF gene. This could be accomplished because Northern Blot electrophoresis utilizes RNA instead of DNA. The RNA of cell types affected with CF, along with the RNA of unaffected cell types were placed on a gel. Probe number two bound to the RNA of affected cell types in the pancreas, colon, and nose, but did not bind to the RNA from non-affected cell types like those of the brain and heart. Probe number one did not bind exclusively to cell types from CF affected areas like probe number two did. From this evidence, it was determined that probe number two contained the CF gene. While isolating the CF gene and screening the genetic library made from mRNA (cDNA library), it was discovered that probe number two did not hybridize. The chances for hybridization may have been decreased because of the low levels of the CF gene present within the probe. Hybridization chances could also have been decreased because the cDNA used was not made from the correct cell type affected with CF. The solution to this lack of hybridization was to produce a cDNA library made exclusively from CF affected cells. This new library was isolated from cells in sweat glands. By using this new cDNA library, probe number two was found to hybridize excessively. It was theorized that this success was due to the large amount of the CF gene present in the sweat glands, or the gene itself could have been involved in a large protein family. Nevertheless, the binding of the probe proved the CF gene was present in the specific sequence of nucleotide bases being analyzed. The isolated gene was proven to be responsible for causing CF by comparing its base pair sequence to the base pair sequence of the same sequence in a non-affected cell. The entire CF cDNA sequence is approximately 6,000 nucleotides long. In those 6,000 n.t.'s, three base pairs were found to be missing in affected cells, all three were in exon #10. This deletion results in the loss of a phenylalanine residue and it accounts for seventy percent of the CF mutations. In addition to this three base pair deletion pattern, up to 200 different mutations have been discovered in the gene accounting for CF, all to varying degrees. The Protein Defect: The Cystic Fibrosis gene is located at 7q31-32 on chromosome number seven and spans about 280 kilo base pairs of genomic DNA. It contains twenty four exons.4 This gene codes for a protein involved in trans-membrane ion transport called the Cystic Fibrosis Transmembrane Conductance Regulator or CFTR. The 1,480 amino acid protein structure of CFTR closely resembles the protein structure of the ABC-transporter super family. It is made up of similar halves, each containing a nucleotide-binding fold (NBF), or an ATP-binding complex, and a membrane spanning domain (MSD). The MSD makes up the transmembrane Cl- channels. There is also a Regulatory Domain (R-Domain) that is located mid-protein which separates both halves of the channels. The R-Domain is unique to CFTR and is not found in any other ABC-transporter. It contains multiple predicted binding sites for protein kinase
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“Every one sees what you appear to be, few really know what you are, and those few dare not oppose themselves to the opinion of the many, who have the majesty of the state to defend them.” - Nicolo Machiavelli, from The Prince Italian political theorist Nicolo Machiavelli speculated that the strongest leaders are ones who are able to carefully balance appearances to his benefit, strategically using them to strengthen his regime. If Machiavelli was indeed correct, then Claudius, from Shakespeare’s Hamlet, starts off as an ideal Machiavellian prince. However, as the play develops, Claudius’ loses his previously immovable command and composure, largely due to his concern over the potential threat posed by his stepson, Hamlet. At the beginning of the play, Claudius appears to have complete control over Elsinore, as evidenced by his imposing speech to the court: Therefore our sometime sister, now our queen, Th’ imperial jointress to this warlike state, Have we (as ‘twere with a defeated joy, With an auspicious and a dropping eye, With mirth in funeral and dirge in marriage, In equal scale weighing delight and dole) Taken to wife... [1.2: 8-14] In this scene, Claudius, who has only recently taken the throne after the death of his brother, addresses some pressing issues. Seeking to create a strong early impression, Claudius uses his words very carefully, taking great pains to both mourn his late brother and celebrate his marriage. Furthermore, with the words “imperial jointress to this warlike state” he justifies the potentially controversial union by making it appear like a benefit to the entire kingdom. Claudius is clearly a shrewd politician, for he deliberately emphasizes the contrast between his marriage and Hamlet’s death, using phrases such as “defeated joy” and “with an auspicious and a dropping eye.” The benefits to such an approach are obvious : on one hand Claudius appeals to popular sentiment by remembering his popular brother, and on the other hand, with his celebration of his marriage, the King proves that he is ready to move on and attack his new role with vigor. The oxymoronic phrases “mirth in funeral” and “dirge in marriage” recall Machiavelli’s words, for Claudius demonstrates his ability to express whatever emotions make him look wise and just, showing that he is in command of Denmark, despite his limited experience as king. Claudius fortifies his majestic appearance by taking decisive and positive action. When faced with the threat of Fortinbras, he immediately takes diplomatic measures, sending Cornelius and Voltemand to protect Denmark’s borders and create an alliance with Norway. Later, Laertes asks for permission to return to France. Knowing the value of the advice of Laertes’ father, Polonius, Claudius gives his consent in a jovial manner, thus strengthening his position with the courtiers. The King even senses the troubled state of Hamlet, and rather than letting things run their course, Claudius immediately sends Rosencrantz and Guildenstern as spies. Most importantly, in every decision he makes, Claudius appears confident, maintaining a balanced temperament in the public eye. Yet underneath this smooth facade lies a man who is concerned above all about Hamlet. A full two months after the death of his father, Hamlet continues to mourn, thereby keeping Old Hamlet’s death in the public spotlight. Claudius, of course, would much rather forget about the incident, for that would not only decrease the likelihood of his being discovered but also help lighten his overburdened conscience. Unfortunately, Hamlet will not let him nor the public forget. Furthermore, Claudius realizes that Hamlet has a justified claim to the throne that could destabilize the King’s regime. In an attempt to alleviate the situation, Claudius stresses Hamlet’s role as his successor, not potential replacement. Nevertheless, the threat of Hamlet remains, and Claudius becomes extremely concerned with it. “That do I long to hear!” [2.2: 53] refers not to news of Fortinbras but to the cause of Hamlet’s perceived lunacy. This exclamation is also the first time that we have seen Claudius stray from his even-tempered public appearance, as he reveals a bit of emotion where Hamlet is concerned. The effect of Hamlet on the King reaches a climax during The Murder of Gonzago, during which the King’s composure breaks down completely. Hamlet’s plan to confirm Claudius’ guilt succeeds brilliantly: when the murder in the play pours poison into Gonzago’s ear, telling the audience that the plot is based on true events, Claudius suddenly rises, shouting “Give me some light. Away!” [3.2: 295] Gone is the calm that had begun to make Claudius a successful leader, replaced by a sudden outburst of emotion in the presence of many others. Now that Claudius’ even-tempered shell has been shattered, we get a better idea of what he would call the “inward man.” [2.2: 6] In the third scene of the third act, we finally see Claudius alone, and he reveals his innermost thoughts while acknowledging his guilt. Clearly, he is not a cold-blooded and inhumane monster but a person whose conscience is making him regret his sins. He explores the similarities between himself and Cain, the Biblical first man to commit fratricide. Claudius knows that in order to achieve divine salvation he must be truly repentant for his sins. However, he is unwilling to give up either the crown or Gertrude, both of which he loves very much, and he resigns himself to a hopeless fate. Claudius is clearly a tormented man who has fallen victim to the temptations of love and power, very similar to the situation of Macbeth. At no point in the play does Claudius glorify his crime; instead, he simply tries to forget about it and move forwards. In the first two acts, Claudius is able to mask his turbulent conscience with a confident appearance. While this approach certainly succeeds in making Claudius a strong leader, it is unable to heal the deep wounds in his soul. As the King wrestles with the increasingly unenviable task of balancing his outward appearance with his interior thought, it is impossible not to feel sorry for him. By the time Claudius kneels and prays, he has been reduced to a man who is now the slave of one terrible deed. To properly portray Claudius, an actor must focus on the gradual fall of the character. In the first two acts, Claudius is at his best, running the court with the sharpness of an experienced leader and decisively acting on every issue of importance. Therefore, the actor must have an imposing and confident presence on stage, for Claudius dominates Elsinore and is in full control of Denmark. However, by the third act, the King must be depicted as a man who is growing increasingly fearful of Hamlet, and during the play, Claudius is so startled that he must appear as though he has seen the ghost of Old Hamlet. But in my opinion, Claudius’ defining moment comes during his lengthy soliloquy in which he acknowledges his guilt. As he mourns his condemned soul, he should seem so helpless that the audience views him with intense pity, for the character of Claudius, like Macbeth, is not intended to represent evil but instead to show the universal ability of power to corrupt and to destroy lives in the process.
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