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The Cystic Fibrosis Gene

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The Cystic Fibrosis Gene


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.2Before 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.3In 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 A and protein Kinase C.4 Mutations in the first MDS are mainly found in exon #4 and exon #7. These types of mutations have been predicted to alter the selectivity of the chloride ion



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