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HASPI Medical Biology Lab 02 Chromosomes are simply portions of DNA wound up and organized into a form that makes it easier for cells to find the directions, or gene, that it needs to make a specific protein. Different organisms have a different number of chromosomes depending on the amount of DNA, or instructions, needed to build and keep that organism functioning. Humans normally have two sets of 23 chromosomes. One set comes from each parent with the same genes, but with different versions of those genes. If they are the same, why do we have two sets? Although each chromosome has the same genes that contain the directions for the corresponding protein, these genes can vary slightly and create the differences we see among humans. For example, the gene for eye color that a child may inherit could be blue, brown, green, or hazel. You will learn more about chromosome structure and inheritance in a later activity. From DNA to Protein Now that you understand that DNA contains the code for proteins, the question becomes how the code in DNA actually leads to proteins? This process is incredibly complex, but can be summarized in three steps: transcription, protein synthesis or translation, and protein folding. Transcription DNA is very fragile and it is vital that not be damaged. For this reason, our bodies have created a way to make a copy of DNA, specifically a gene, so that it doesn’t have to leave the protection of the nucleus. The copy is made out of RNA, or ribonucleic acid, called messenger RNA, or mRNA. This copying process is called transcription and ONLY occurs in the nucleus. Protein Synthesis or Translation Once an mRNA copy of the gene has been created, the ribosome can build a protein using the mRNA copy as directions. The ribosome translates the order of amino acids in the protein and bonds them together into a chain. Protein Folding The length of the amino acid chain produced by ribosomes can range from only a few hundred to hundreds of thousands of amino acids long. The amino acid chain is transported Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 1 to the endoplasmic reticulum (ER) where they are folded and can even have carbohydrates or lipids added to them to produce functioning proteins. An amino acid chain cannot perform a function until it has been folded into its functional shape. Amino acid chains are also known as polypeptide chains. The interactions and bonds that occur between the different amino acids are what cause the folding and shaping of the protein. Every amino acid has a functional side that can cause or prohibit bonding with other amino acids. Proteins, called chaperones, can assist an amino acid chain during the folding and bonding process to create a finished protein that can now perform a function in the body. Cystic Fibrosis Cystic fibrosis is a common genetic disease caused by a mutation in a gene called the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene is located on chromosome 7 and has the directions to create the CFTR protein. The CFTR protein is a channel protein that regulates how salts, most commonly sodium (Na+) and chloride (Cl-), and water move through the cell membranes of epithelial cells. Epithelial cells cover surfaces of the body and can be found in the skin, respiratory, and digestive tracts. Na+ and Cl- help control the movement of water into tissues. When the CFTR protein does not function correctly, chloride (Cl-) is unable to pass through the center channel and sodium (Na+) is also unable to pass through the cell membrane. When they are imbalanced, watery substances like mucus are unable to move into the tissues and the mucus becomes extremely sticky and thick. (The role of mucus is to lubricate the surfaces of the body.) As a result, symptoms of cystic fibrosis include: Extremely salty skin Thick, sticky mucus that can block respiratory and digestive tracts Frequent respiratory infections due to bacteria trapped in mucus Wheezing, persistent cough, and shortness of breath Lack of digestion leading to poor growth/weight The cystic fibrosis mutation is a recessive disorder passed from parent to offspring. This means an individual needs two copies of the mutated CFTR gene to have cystic fibrosis. Since the mutation is recessive, a parent may not have symptoms of cystic fibrosis or know they carry the mutation. There are more than 30,000 people in the U.S. with cystic fibrosis and more than 1,000 cases are diagnosed yearly. More than 10 million people in the U.S. are carriers of cystic fibrosis. When cystic fibrosis was first discovered, few sufferers lived past 6 years old, but due to medical advances the median age of survival has increased to 37 years old. 2 Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 Review Questions 1. What are proteins? Give 3 examples of functions they perform. (see “Macromolecule” notes) 2. How are DNA, genes, chromosomes, and proteins related? 3. Describe protein folding. What causes an amino acid chain to fold? (see “More on Protein” notes) 4. What is a mutation? Explain how a mutation in a gene can influence the protein it creates. 5. List and explain 2 types of mutations. 6. What is cystic fibrosis? List 3 symptoms associated with cystic fibrosis. 7. What is the purpose of the CFTR protein? 8. What happens when the CFTR protein is mutated? 9. How does an individual get cystic fibrosis? Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 3 Purpose Create a model to simulate the process by which a protein is produced, and how a mutation can impact a protein’s function. Background DNA contains the directions to create the proteins that allow our bodies to function. Portions of the DNA that contain directions for a single protein are called genes. Because DNA is delicate we DO NOT want to remove it from the nucleus and instead it makes a copy of the directions using RNA. RNA polymerase is a protein that copies the DNA and the finished copy is made of messenger RNA, or mRNA. This copying process is called transcription. After transcription, the copy leaves the nucleus and is in the nucleus with special organelles called ribosomes. The ribosomes translate the directions from the copy to build a protein in a process called translation. Lastly, the protein must fold into its final shape in order to be able to perform a function. In this activity, your team will be making a copy of the CFTR gene that contains the directions for creating the CFTR protein. This protein is a transport protein that embeds itself in the cell membrane and regulates certain substances (Na+ and Cl-) moving in and out of the cells in the skin, pancreas, and lungs. If this protein is not built correctly, and therefore not able to function correctly, these substances cannot move in/out of the cell and cause a thickening and build-up of mucus. This causes the condition known as cystic fibrosis. Procedure/Directions Your lab team will be given tasks, or directions, to perform on the left. Record your questions, observations, or required response on the right when space is available. Part A: Set-Up Task Response Obtain the following supplies: 1 4 Scissors Tape Normal CFTR Gene sheets (2 pgs) Mutated CFTR Gene sheets (2 pgs) Blank white paper for mRNA (transcription) 2 RNA Polymerase sheets (1 pg) 2 Ribosome sheets (1 pg) Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 2 3 4 Cut out the “Normal CFTR Gene” and “Mutated CFTR Gene” strips along the dotted line and tape each end together. Make sure to tape the correct ends to one another to create a single DNA strand of the normal CFTR gene AND the mutated CFTR gene (see image.) You will transcribing the mRNA on the blank white sheets of paper from your supplies you’ve obtained. Turn the paper landscape and cut this paper into one inch strips. You will be taping these strips together using the amount of strips it takes to transcribe the DNA. The “tRNA templates” have been cut out for you and are in the bowls of beads that correspond to the correct amino acids. (The beads are the amino acids) The RNA Polymerase and Ribosome templates have been cut out for you. 5 Part B: Transcription Task 1 Get into a team of 4. Within your team separate into pairs. One pair will be following the procedure with the “Normal CFTR Gene” and the other pair will be following the EXACT same procedure with the “Mutated CFTR Gene.” 2 In this part of the activity, your team will be transcribing the normal and mutated CFTR gene that contains the directions for creating the CFTR protein. Response Mutated Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 Normal 5 a. Where does transcription occur? 3 RNA polymerase is the protein that functions to unzip, or open, the DNA double helix and bond RNA nucleotides as they match up to the DNA nucleotides creating the mRNA strand. Remember Cytosine bonds to Guanine and Adenine bonds to Thymine. In RNA, Thymine becomes Uracil. 4 a. What are the 4 DNA nucleotides? b. What are the 4 RNA nucleotides? c. Which nucleotides complementary base pair with one another in DNA? In RNA? 1. DNA: 2. RNA: 5 6 7 8 6 Place your “RNA Polymerase” template on the table (each pair of team members need a template). Feed the START end of your CFTR gene into and through the cut in the RNA polymerase sheet (see image). Starting at the START end of the CFTR gene, place the blank white paper strips next to the CFTR genes. On the white paper strips, write the correct RNA nucleotides that base pair with the DNA nucleotides. You are creating your mRNA strand! For example, the Adenine DNA nucleotide will match with a Uracil RNA nucleotide. As you move down the CFTR gene, slide it through the RNA polymerase. Continue this process of copying the DNA nucleotides matching RNA nucleotides, as you write them on the white paper strips (mRNA strand), until you reach the end of the CFTR gene. Tape your white paper strips together as you run out of room completing each strip. Make sure to tape them in order! Remove the CFTR gene from the RNA polymerase. You have completed an RNA copy of the CFTR gene. This copy is called messenger RNA, or mRNA. In an actual cell, this process would be repeated with the same CFTR gene and RNA polymerase many times, to create multiple mRNA copies. Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 a. What is the function of RNA polymerase? b. What is mRNA? Why is it important to create mRNA rather than use actual DNA for the next step? c. Summarize the transcription process. Part C: Translation Task 1 2 3 Response In this part of the activity, your team will be using the mRNA copy of the CFTR gene you created in transcription to decode the order of amino acids that make up the CFTR protein. a. What is the purpose of translation? To simulate the structure of proteins, you will be using plastic beads to represent the 20 amino acids that make up protein. The plastic beads (amino acids) are in the glass jars on the back counter. Before you build your protein by collecting the amino tRNA mRNA acids, place your “Ribosome” on the table. Feed your mRNA copy into the ribosome with the written nucleotides facing up. Only the first three mRNA nucleotides should be visible in the window of the ribosome. rRNA Note: Your mRNA is hand written on the white paper strips and does not look the the mRNA in this picture; the tRNA’s in this picture are in the bowls of plastic beads (amino acids) Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 7 4 5 6 ! ! ! ! ! ! Phenylalanine Phenylalanine Leucine ! ! ! ! ! ! tRNA tRNA tRNA ! ! ! Every three mRNA nucleotide bases are called a codon. Each codon is actually a 3-base code A A ! A ! A A A A G ! for a specific amino acid. There are 61 possible codons means ! that code for 20 ! amino acids. This ! ! some amino acids have more than one codon. ! ! ! ! ! ! ! ! ! ! ! ! Using your mRNA codon chart, write the correct amino! acid under each codon on your ! ! mRNA ! ! ! strand (white paper strip). Leucine Leucine Leucine ! ! ! ! tRNA ! tRNA ! tRNA ! ! ! Obtain two pipe cleaners A A A G ! G A ! G ! G ! ! ! ! Transfer RNA (tRNA) float around in the cytoplasm near! ! ! ! ! ribosomes and the endoplasmic reticulum. One end of! ! ! ! ! ! ! a tRNA molecule has an amino acid attached and the! ! ! ! ! ! other end has a set of 3 bases that can match up to an Isoleucine Isoleucine Isoleucine ! ! ! mRNA codon. This 3-base code located on tRNA is ! ! ! tRNA tRNA tRNA ! ! ! called the anticodon. A A A G ! U A ! U ! U ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Using your amino acid sequence you wrote on your mRNA strand below the ! ! the codons, locate ! ! ! corresponding beads and string them onto your pipe cleaner in the correct order. See !step 9 Valine Valine Valine ! ! ! below before you start! ! ! ! tRNA tRNA tRNA ! ! ! A A A G ! C A ! C ! C For example, the first mRNA codon is “AUG” so the anticodon is “UAC”,! which codes for! the ! ! methionine amino acid. C U amino acid 7 U anticodon 8 9 Slightly bend the end of the pipe cleaner to prevent the amino acids/beads from sliding off the end. Place the amino acid/bead on your pipe cleaner and slide it to the end (see image). Continue this process until you reach the end of the 10 mRNA copy and hit a STOP codon. Fold the end of the pipe cleaner to prevent the amino acids/beads from falling off. You have just created an amino acid chain. a. Summarize the translation process. 8 Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 U ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Part D: Protein Folding Task 1 Response So far you have made an mRNA copy of the CFTR gene and decoded the copy to determine the amino acid order. You have an amino acid chain, but it is not yet a functional protein. Different amino acids interact and bond with each other causing the amino acid strand to fold and create a protein that can perform a function. http://www.piercenet.com/media/ProStructureFig1.gif glue dot 2 Obtain a glue dot sheet (share among your team). Place a glue dot in each of the 6 spaces of the methionine amino acid/bead (see image). glue dot glue dot glue dot glue dot glue dot 3 4 For your amino acid strand, methionine and glycine will bond to each other. (both are clear in color) Starting at methionine, follow the amino acid chain until you find a clear bead (glycine). Fold the amino acid strand and connect glycine to a space in methionine at one of the glue dots (see image). methionine glycine 5 Continue down the amino acid chain, folding and connecting any glycine to the next open space on methionine, until all of the glycine amino acids on the chain have been attached. Mutated 6 You now have a completed protein! Normal Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 9 Part E: Protein Function Task 1 Now that your team has completed a normal and mutated CFTR proteins, compare their structures. 2 CFTR functions to move salts in/out of cells. You have only created a portion of this protein that attaches and anchors the remaining protein to the cell membrane. The complete CFTR protein is actually more than 1,300 amino acids long! 3 The part of the normal CFTR protein that you created is responsible for connecting the CFTR protein to the cell membrane. The green portion of your normal protein would be the active site that binds to a portion of the cell membrane. 4 Your instructor has taped up a simulated cell membrane. Take your normal and mutated CFTR proteins to the simulated cell membrane and see if the green active site binds and stays attached to the green attachment point on the cell membrane (see figure to the right) Response a. Compare and contrast the structure of your normal and mutated CFTR proteins. Med Bio Lab 02: The Cell Membrane The cystic fibrosis transmembrane conductance regulator (CFTR) protein creates a channel for chloride ions (Cl -) to move through the cell membrane of epithelial cells lining the lungs, pancreas, skin, and other surfaces of the body. · NORMAL PROTEIN: If your CFTR protein is normal and attaches the cell membrane, it will allow Cl – to move through the cell membrane and maintain a balance of ions on the outside and inside of the cell. As a result of this balance, mucus within the airways hydrated and functioning correctly. · MUTATED PROTEIN: If your CFTR protein is mutated and DOES NOT attach or open Cl – cannot move through the cell membrane. This creates an imbalance in the amount of ions on the outside and inside of the cell membrane and affects the amount of water in the cell. As a result, mucus becomes extra sticky and is difficult to remove from the lungs. This includes any bacteria trapped in the mucus, which can cause numerous respiratory infections. This is only ONE example of a symptom of cystic fibrosis. ! ! ! ! Cl#$# ! Outside Lung Airway ! 5 10 You have completed this activity! DNA RNA protein is known as the central dogma of genetics, and you now have first-hand experience as to how this process happens. In actuality, your cells can transcribe a gene that is 1,500 base pairs and produce the protein it codes for within 1.2 seconds! Cl#$# Cl#$# Cl#$# Cl#$# Cell Membrane Inside Lung Airway mucus! mucus! Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 mucus! Analysis & Interpretation Analysis Questions 1. Compare and contrast how the normal and mutated proteins adhered to the cell membrane? How did the mutation impact the function of the CFTR protein? 2. What happened to the green active site (place where the reaction takes place) of the protein in the mutated CFTR protein? Why is this a problem? 3. Hypothesize what might happen if the deletion mutation was at the end of the gene rather than towards the beginning. 4. Explain how the CFTR gene leads to the creation of the CFTR protein? 5. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins, which carry out essential functions of life through systems of specialized cells. Genes, Proteins, and Disease; HASPI Medical Biology Lab 02 11