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Gene Essay, Research Paper

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 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 channels.4

Mutations that are in the first NBF are predominant in

CFTR. As previously mentioned, 70 percent of the mutations

arising in CF cases are deletions of three base pairs in

exon #10. These three base pairs give rise to phenylalanine

and a mutation at this site is referred to as DF508.5 Such

a mutation appears not to interfere with R-Domain

phosphorylation and has even been reported to transport

chloride ions.6&7

There are five other frequent mutations that occur in the

first NBF. The first is a deletion of an isoleucine

residue, DF507. The second is a substitution of glycine or

amino acid #551 by aspartic acid/F551D. The third involves

stop mutations at arginine #553 and glycine #542. The

fourth is substitutions of serine #549 by various other

residues. The fifth is a predicted splicing mutation at the

start of exon #11.7

Mutations within the R-Domain are extremely rare. The only

reason they do occur is because of frameshifts. Frameshifts

are mutations occurring due to the starting of the reading

frame one or two nucleotides later than in the normal gene

translation.4

Mutations in the second membrane spanning domain of the

CFTR are also very rare and have only been detected in exon

#17b. These have no relevance to mutations occurring in the

first membrane spanning domain. They apparently do not have

a significant impact on the Cystic Fibrosis Transmembrane

Conductance Regulator either.4

Mutations in the second nucleotide-binding fold occur

frequently in exon #19 and exon #20 by the deletion of a

stop signal at amino acid number 1282. Exon #21 is

sometimes mutated by the substitution of asparagine #1303

with lysine #N1303K.4

The Bio-Chemical Defect:

Studies of the chloride channels on epithelial cells lining

the lungs, sweat glands, and pancreas have shown a consensus

in that the activation of chloride secretion in response to

cAMP (adenosine 3′, 5′-monophosphate) is impaired in cystic

fibrosis cases. Another affected, independently regulated

chloride channel that has been discovered is activated by

calcium-dependent protein kinases. Sodium ions have also

been noted to be increasingly absorbed by apical sodium

channels.8 Therefore, the lack of regulated chloride ion

transport across the apical membranes and apical absorption

of sodium ions, impedes the extracellular presence of water.

Water will diffuse osmotically into cells and will thus

cause the dehydration of the sol (5- mm fluid layer of the

cell membrane) and the gel (blanket of mucus) produced by

epithelial cells.9 As a result of this diffusion of water,

airways become blocked and pancreatic proteins turn

inactive.

An Account of the Absorption and Secretion of Cl-, Na+, and

Proteins:

An inward, electrochemical Na+ gradient is generated by the

Na+, K+-ATPase pump located in the basolateral membrane (the

cell side facing the organ it is lining). A basolateral

co-transporter then uses the Na+ gradient to transport Cl-

into the cell against its own gradient. This is done in

such a way that when the apical Cl- channels within the

membrane spanning domain open, Cl- diffuse passively with

their gradient through the cell membrane.4

In pancreatic duct cells, a Na+, H+-ATPase pump is used and

a bicarbonate secretion is exchanged for Cl- uptake in the

apical membrane. Chloride ions then diffuse passively when

the Cl- channels are opened. Such secretions also allow for

the exocytosis of proteins in the pancreas which will later

be taken into the small intestines for the breaking down of

carbohydrates.4

In addition to the pump-driven gradients and secretions,

there exists autonomic neurotransmitter secretions from

epithelial cells and exocrine glands. Fluid secretion,

including Cl-, is stimulated predominately by cholinergic,

a-adrenergic mechanisms, and the b-adrenergic actions.4

Such chemical messengers cannot enter the cell, they can

only bind to specific receptors on the cell surface and

transmit messages to and through an intracellular messenger

such as Ca2+ and cAMP by increasing their concentration.

The intracellular message is transmitted across the cell by

either diffusion or by a direct cascade. One example of a

directed cascade is the following:

Possible Treatments For Cystic Fibrosis:

One suggested treatment for CF has been to provide the

missing chemicals to the epithelial cells. This can be

accomplished by the addition of adenosine

3′,5′-monophosphate (cAMP) or the addition of the nucleotide

triphosphates ATP or UTP to cultures of nasal and tracheal

epithelia. This has been proven to alter the rate of Cl-

secretion by removing the 5-mmeter sol layer of fluid in the

respiratory tract.9 Moreover, luminal application of the

compound amiloride, which inhibits active Na+ absorption by

blocking Na+ conductance in the apical membrane, reduced

cell secretion and absorption to a steady state value.

Another treatment that has been suggested is to squirt

solutions of genetically engineered cold viruses in an

aerosol form into the nasal passages and into the lungs of

people infected with CF. This is done in hopes that the

virus will transport corrected copies of the mutated gene

into the affected person’s airways so it can replace the

mutated nucleotides.10 This form of treatment is known as

gene therapy.

A different approach taken in an attempt to cure cystic

fibrosis involves correcting the disease while the affected

“person” is still an embryo. Test tube fertilization (in

vitro fertilization) and diagnosis of F508 during embryonic

development can be accomplished through a biopsy of a

cleavage-stage embryo, and amplification of DNA from single

embryonic cells.5 After this treatment, only unaffected

embryos would be selected for implantation into the uterus.

Affected embryo’s would be discarded.

Conclusion:

Chloride conductance channels have dramatic potentials.

One channel can conduct from 1×106 to 1×108 ions per

second.8 This is particularly impressive when you consider

the fact that there are not many channels present on cells

to perform the required tasks. As a result of this, a

mutation of one channel or even a partial mutation of a

channel, that causes a decrease in the percentage of channel

openings, can exert a major effect.

Even the mildest of cures altering the Cystic Fibrosis

Conductance Regulator in CF afflicted people would lead to

significant improvements in that individuals health. Since

cystic fibrosis is the most common genetic disorder,

particularly amongst Caucasians, in today’s society, intense

research efforts towards its cure would be invaluable. When

will cystic fibrosis be completely cured? No one can say

for sure but, strong steps have already been taken towards

reaching this goal.

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