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