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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.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 A and protein Kinase C.4

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