Sequencing the Cystic Fibrosis Gene:
1994 Woodrow Wilson Collection
Many genetic disorders can be identified by analyzing DNA obtained from white blood cells. Studies may also be carried out on cells obtained from a fetus via amniocentesis. Cystic Fibrosis (CF) is such a disorder. It is recessive, is carried on chromosome 7, and mainly affects Caucasians. CF was recognized as early as the seventeenth century when it was known that " a child that is salty to taste will die shortly after birth." Other symptoms include small body size, severe respiratory congestion, stubby fingers and foul smelling stools. In 1989 Francis Collins and his colleagues located the gene and identified the defect that causes this condition. The mutation is a result of the omission of a single amino acid, phenylalanine. This is caused by an error at codon 508, one of 1480 codons. The normal protein forms a channel in the membranes of cells that line the respiratory passages, this channel acts as a passageway through which sodium ions enter and chloride ions leave cells. The defect in the CF protein prevents chloride ions from causing cells to retain water. The consequence of this is a formation of thick mucus that surrounds the cells of an affected individual.
This activity permits students to simulate the sequencing of a gene and to detect the presence of possible genetic defects. Students will use diagrams that represent bands of nucleotides in order to determine the sequence of a small piece of the CF gene. Comparisons will be made between base sequences of pairs of chromosomes from individuals who are not affected by CF and those who are. Through the use of autoradiographs* and gel electrophoresis, it is possible to separate the bases and reveal their sequence. Some sequences are variations that lead to the production of proteins which have the same amino acids and therefore no genetic disorder. Other sequences consist of triplets that code for different amino acids and lead to the production of an abnormal protein that may produce cystic fibrosis. In order to test for a recessive genetic disorder such as CF, the alleles from both parents must be examined.
*Autoradiographs are used to determine the sequence of nucleotides in a strand of DNA. The sequence is usually read from the bottom to the top. For the sake of convenience, the bases in this activity will be read from top to bottom.
Figure 1.4 lists the DNA sequences and amino acids for the maternal and paternal chromosomes of four individuals. Josina is homozygous normal, her DNA sequences and amino acids should be used as a basis for comparison. It is suggested that students come to this conclusion on their own. Leah has CF; her maternal and paternal chromosomes have slightly different sequences; both result in the substitution of serine for phenylalanine. Martin is heterozygous normal; he has received an abnormal sequence from his mother, but does not have CF because he has a normal chromosome from his father. Richard has CF; an entire codon is missing on both the maternal and paternal chromosome, resulting in the loss of the amino acid phenylalanine.
- The student will use laboratory data in order to determine nucleotide sequences.
- The student will identify those base sequences that are normal, those that are normal variants and those that cause cystic fibrosis.
- The student will use DNA nucleotide sequences to determine amino acid sequences.
9th grade biology
One 50 minute period
One copy each of the Student Worksheet and Student Material
BSCS and the American Medical Association. Mapping and Sequencing the Human Genome: Science, Ethics and Public Policy. 1992.
Howard Hughes Medical Institute. 1991. Blazing a Genetic Trail.
Kerem, B.S., et al. "Identification of the cystic fibrosis gene: genetic analysis." Science, 245:1073-1080, 1989
Lewis, Ricki. Human Genetics. Wm. C. Brown Publishers,1994.
Riordan, J.R. et al. "Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA." Science, 245: 1059-1065, 1989.
Rommens, J.M., et al. "Identification of the cystic fibrosis gene: Chromosome walking and jumping." Science, 245: 1059-1065, 1989.
Karen Armstrong-Malatesta, Ph.D. Senior Lecturer, Director, Introductory Laboratories, Princeton University. Autoradiograph.
- Cystic Fibrosis Foundation
6931 Arlington Road
Bethesda, MD 20814
- Cystic Fibrosis Research Trust
5 Blyth Rd.
Bromley, Kent BR1 3RS
- For information on genetics and genetic counseling referrals, please contact:
- March of Dimes Birth Defects Foundation
1275 Mamaroneck Ave.
White Plains, NY 10605
- Alliance of Genetic Support Groups
35 Wisconsin Circle, Suite 440
Chevy Chase, MD 20815
(800) 336 -GENE
Cystic Fibrosis (CF) and Sickle Cell Anemia are diseases inherited in a similar manner. Both diseases are recessive and caused by a change in an amino acid at a specific position on a protein. CF is caused by a change in a protein found in cell membranes and Sickle Cell is caused by a change in the pigment called hemoglobin found in red blood cells. While Sickle Cell Anemia mainly affects African-Americans, CF affects mainly Americans of Northern European descent. The most common complaints associated with CF are severe problems with respiration and digestion as well as very salty sweat. These symptoms are caused by cells being unable to pass sodium and chloride through the cell membrane. This situation explains the salty sweat.
CF may cause more severe problems as the patient gets older. Older victims may have more difficulty breathing and digestive problems may increase. The digestive condition may be treated by taking the enzymes which need to replaced. Adults may suffer from nasal and cardiac problems, mild diabetes and may experience massive bleeding from the bronchial tubes. Most males who have CF are unable to have children because of damage to small tubes in the testes. Females may also experience fertility problems.
In order to locate the CF gene, geneticists used a linkage map to identify which chromosome carried the gene. The scientists searched for identifiable pieces of DNA on specific chromosomes by comparing the DNA of individuals afflicted with CF to the DNA of their parents, relations and other afflicted individuals. After many years of searching, scientists determined that the gene for CF was located on chromosome number 7. The gene map of this chromosome indicated to the researchers the exact location of each piece of DNA in the CF gene. Next the base sequences of individual pieces of DNA were examined in order to find the cause of the abnormality in the CF gene. Approximately 70 percent of the people who have Cystic Fibrosis exhibit the same mutation in their DNA nucleotide sequences.
In this activity a short sequence of both maternal and paternal DNA will be examined in order to determine a few types of nucleotide changes that may cause CF.
- One method employed by scientists in the search for the CF gene is the use of radioactivity in order to detect DNA on a piece of photographic film. Examine Figure 1.1.
This is an autoradiograph. Each blot on the film indicates one of the four nucleotides. Each base is found in its own column. Reading from left to right, a blot in the first column indicates that the nucleotide is adenine; a blot in the second column indicates that the nucleotide is cytosine; a blot in the third column indicates that the nucleotide is guanine; and a blot in the fourth column indicates that the nucleotide is thymine. Determine the base sequence for the top ten positions. The correct sequence is A,C,G,G,G,G,C,G,T,T. This is one method used by geneticists to find the sequence of nucleotides in a piece of DNA.
- Examine Figure 1.2. This figure represents autoradiographs similar to the one shown is Figure 1.1. Each strip represents a small number of the bases located on the gene for CF from the four named individuals (these are from the maternal chromosome). In the column marked DNA BASE, list the sequences of bases from the maternal chromosome for each of the named individuals.
- Carefully transfer the base sequence for each individual to the student worksheet. Check your work by comparing your worksheets with your lab partner.
- Examine the base sequences of all four individuals and circle any differences. Do any of the individuals have the same sequence in both the maternal and paternal chromosome? If so, name the individual. Is this person said to be homozygous or heterozygous?
- Examine Richard's base sequences. What is different about his DNA? Propose a hypothesis to explain your observations.
- CF is caused by a defective protein. The instructions for making a protein are contained in the base sequences of DNA. Using Figure 1.3 (below) and the DNA sequences you determined from the autoradiographs, fill in the column marked amino acid for each individual on Fig. 1.4. Be sure to consider the maternal and paternal sequence together for each individual. If the sequence is not the same, check the DNA coding chart carefully. Does a difference in base sequence always mean a difference in the amino acid? Explain your answer.
- If the difference in base sequence is drastically different, compare the sequence to Josina's DNA. Josina is homozygous normal. Remember, CF is homozygous recessive. Therefore, if one chromosome has a sequence exactly the same as Josina's, the individual is heterozygous and does not have CF but does carry the gene. Who is heterozygous for CF?
- How does Leah's amino acid sequence differ from Josina's? Do you think Leah has CF? Why or why not?
- Compare the amino acid sequence of Richard with Josina's. What is the defect in Richard's amino acid sequence? What accounts for this?
Diagram of chromosome #7
Teacher Answer Sheet -- DNA sequences and amino acids