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Gene Expression Here is genetics in a nutshell: DNA RNA protein DNA RNA is called Transcription, occurs in nucleus RNA protein is called Translation, occurs in the cytoplasm, uses free or bound ribosomes. Proteins are the workhorses of the body. They are responsible for our physiology. You should not denature them. The primary structure of a protein is the string of amino acids. All AA’s have the following things in common: 1. A central carbon 2. A carboxyl group (which is acidic, because it is an electron acceptor and hydrogen donor) 3. An amine group (an electron buzzing around nitrogen. This means it can donate an electron). Amine groups are basic since they remove hydrogen and donate an electron. 4. They all have at least one hydrogen coming off the central carbon. AAs differ in their R group (called the functional group). The R group dictates their chemistry and is what makes each type of AA different from the others. The R groups interact with each other as well as other things in the cell to create the physiology. You make a protein by combining several AA’s in process called dehydration synthesis. The monomer (individual AA) has now become a dimer (two AAs). Keep combining AAs you get a polypeptide. They are linked by peptide bonds. There are 20 essential AA’s don’t memorize them. On each one, find their central carbon, hydrogen, carboxyl group, and amine group. Which one has an extra carboxyl group? Glutamic acid. If a protein is made up of many Glutamic acids, it will be an acidic protein. Find two AAs with an R group that has extra amines (Arginine and Lysine). These extra amine groups act like a base because they bind with hydrogen in a solution. AAs with benzyl rings, such as tyrosine, don’t like water, so tyrosine will be hydrophobic. If a protein made of a lot of tyrosine is a hormone, it will be secreted into the blood, where it dissolves in the water there. It will not be easy for that protein to travel around in the plasma; it will need a carrier protein to help it. An example of this type of hormone is thyroid hormone. Histidine is a great AA. What does it have on its side chain? An amine group, except the group is in a ring structure. A protein like this is critical to maintain acid-base balance. They can both bind and release hydrogen in solutions, to adjust the pH as needed. Denaturing proteins leads to loss of function. When a protein is properly folded in its tertiary structure, let’s say that three particular AA are close to each other. Those three R groups need to do a particular job on the substrate. For an analogy, say the substrate is an orange, and the R group is the juicer. If the protein is unraveled, the orange cannot be turned into orange juice. We need the tertiary structure of the protein to be intact. Proteins can be denatured by heat, like egg yolk that goes from clear liquid to solid white. As the proteins unravel, they get larger and precipitate out of solution, so they solidify. pH and salts can also denature proteins. When you cure meat with salt, it eliminates bacteria proteins as well, so the meat stays preserved a long time. A primary protein structure can be joined to itself by sulfide bridges, H bonds, and hydrophobic interactions to help wrap it into a tertiary structure. A protein cannot fold correctly if a single AA is changed. This defect is caused by a gene defect. Change a single AA and it cannot interact like it did before. If Glutamic acid (acidic) was replaced by another acidic acid, the protein might work okay. It might cause the protein to wrap around itself a little more loosely, and who knows? It might work even faster. Even if it works slower, it still probably will work okay. But if substituted by basic AA, can be a big problem. Valine causes a kink in a protein. If the mutation substitution is Valine, the result is usually a big problem. Loss of a Valine where there should be one is also a problem because a kink is not where it should be. A mutation can be harmful, beneficial, or cause no change. The more similar a substitution, the less of a problem. But there is always a change in physiology. Ten percent of all breast cancers are congenital genetic mutations (the woman is born with it), the rest are from a spontaneous mutation of the gene. It gives us a way to study that cancer. Brca 1 is a breast cancer genetic mutation. Genes code for proteins that carry out functions, as well as proteins that regulate the functional proteins. It tells the functional protein where to go or what to do. For example, one type of protein that is made in the cytoplasm is supposed to return immediately to the nucleus to perform its function. It knows to do this because its regulatory protein gives it instructions. If a mutation occurs in the gene so the functional protein is made but the regulatory gene is not, the functional protein will be there, but it will not know return to the nucleus to perform its job. The functional protein is there, but not in the right place. Genes code for proteins, and are the functional units of DNA. DNA is a double stranded string of nucleic acids, twisted together in a helix, like a twisted ribbon. Take a cheek cell out of your mouth and add salt to it, and it will thicken up and look like snot. This is because the salt ruptured cell and released the DNA and you are now handling the DNA. Magnify it and can see billions of base pairs of DNA nucleotides in one cell. How do they come together to make a gene? You have 27 thousand genes with known functions, the rest are called junk, but we just don’t know what they do (they probably regulate the other proteins). A cookbook is like a genome. A cookbook is a collection of recipes, an index, plus tips on how to measure, convert to metric, etc. In the DNA you also have a series of recipes, or genes. There are other things in a cookbook too, including blank spaces, or other junk. If you delete junk genes from a mouse, there should be no change in the mouse, but in experiments, they found that the mouse did have problems. Cookbooks have recipes for chocolate chips, marinara sauce, fish dishes, pies, but each cookbook has different recipes. We all have a recipe for hair color, but we each have different color hair. Some have strong fingernails, some have weak. These different recipes are organized in the same way, with spaces between recipes. If a section of a gene does not create a protein, they call it junk. But it is not a good name, because those genes do something, we just don’t know what they do. Likewise, if you stop after making just the cookie dough, there would be no cookies. We need those genes to do things other than code for proteins. A recipe tells you what order to apply the ingredients and how to mix them. Some of these junk DNA segments do have a function: to regulate the gene expression of other genes. There are proteins whose job it is to monitor the cell size, and when the cell gets too big, they race back to the nucleus to tell the cell to divide. A gene codes for those proteins: proto-oncogenes. They are supposed to be there, maintained in homeostasis by other proteins. Those genes come from genes too, tumorsuppressor genes. That means that we have genes that encourage cell division (proto-oncogenes) and genes that discourage cell division (tumor-suppression genes). Proto-oncogenes and tumor suppressor genes should be in same amount. If the proto-oncogene is made but not the tumor suppressor, there will be more cell growth and division. What if the mutation caused excess proto-oncogenes to form, but the person had a normal amount of tumor suppressors? You would also get a tumor. In DNA, nucleotides pair with each other. When the DNA unravels to make an RNA template, the DNA nucleotide “T” pairs to the RNA nucleotide “A”, etc. This pairing process is transcription. When the newly formed RNA strand detaches itself from the DNA template and floats out of the nucleus, a second RNA can pair with the first RNA, blocking it from binding to the ribosome, so it cannot make a protein. Therefore, in a patient that has a mutation in a proto-oncogene that favors too much cell division, you can inject this special type of blocking RNA into the patient, and the injected RNA blocker goes to the RNA of the proto-oncogene and binds to it so the proto-oncogene cannot be made, and the cancer will stop! How does the injected RNA know where to go in the body? Scorpion venom loves RNA, and heads right to it in every cell, so we combine the venom with the injected RNA and it takes it to the right place! What if you want to turn on a tumor suppressor gene? Certain “egg RNA” will bolster this. You can inject an RNA egg. Suppose that a good RNA has been shut down by a bad RNA blocker. Inject the RNA egg to bind with the bad one so the good one is left alone so that it can bind with the ribosome and make the protein. This happens in the body naturally, but we are trying to make it in large amounts to inject. Mitochondria have their own DNA, RNA, proteins, and there is more research being done there, as well. Brain surgeons want to take out tumors surgically before chemotherapy is begun. They don’t want to take good tissue, so how much tissue do you take before you accidentally scrape out 10 years of piano lessons? Sprinkle scorpion venom on the patient’s brain during the surgery, when the skull bone has been removed, turn on a black light overhead, and wherever it lights up, scrape that out. This can be done for prostate and breast cancer, as long as the involved tissues are superficial. This type of medicine is called Molecular medicine, very interesting. The ultimate website (not Wikipedia or Web MD) to learn about the latest things in molecular medicine is here: www.nlm.nih.gov CLASS DEMONSTRATION OF PROTEIN SYNTHESIS Need four groups of students with 10 people in each group: 10 students with black shirts (represent the nucleotide Adenine “A”) 10 students with white shirts (represent the nucleotide Thymine “T”) 10 students with blue shirts (represent the nucleotide Cytosine “C”) 10 students with grey shirts (represent the nucleotide Guanine “G”) The copying of the original chromosome is during transcription. In this process, think of wanting to make a duplicate of your hand. You first take a plaster impression of your hand print. This is transcription, but it does not give you a copy of your hand, it just makes a negative cast of your hand. The negative cast is the RNA strand. During translation, the RNA is copied. In this process, we take the negative cast of your hand and fill it will plaster. Then we peel away the negative cast, and are left with the plaster positive cast, which is the duplicate of your hand. When the DNA unravels to make an RNA template, the DNA nucleotide “T” pairs to the RNA nucleotide “A”, etc. This pairing process is transcription. The newly formed RNA strand detaches itself from the DNA template and floats out of the nucleus, into the cytoplasm. There, it binds to a ribosome, which reads the RNA strand and attaches a "T" nucleotide to the RNA "A" nucleotide, etc, until the entire RNA strand has been paired with nucleotides. This is translation. Then the RNA strand is taken away, taken apart, and its nucleotides are recycled. The string of nucleotides that is left is then read in a different way: Every three nucleotides codes for one amino acid. When the ribosome finishes reading all the nucleotides and forming a string of amino acids, that is the protein. The first task in protein synthesis (gene expression) is to make a strand of DNA. One of each of the students will stand next to each other in this sequence: Grey, white, white, blue, black, blue, grey, grey, grey. Each nucleotide should be bound to the next one by dehydration synthesis, so now hold hands. This represents one of the DNA strands in one of our chromosomes. To make the second strand of the double helix of DNA, a Black shirt (A) will base pair with a white shirt (T) and a blue shirt (C) will base pair with a grey shirt (G). Someone with a blue shirt must go and face the first person in out sequence, who is wearing a grey shirt. Then 2 black shirts are needed, then a grey, etc. This second strand also needs to be bound together by dehydration synthesis, so hold hands. To complete the double-stranded DNA, we need hydrogen bonding between the two strands, so those in the first strand should put their toes on the toes of the person in the second strand, who are facing them. Now we have a segment (gene) of our double stranded DNA chain, and we are still located in the nucleus. If DNA leaves the nucleus, it will be targeted for destruction. Therefore, if we want to make a copy of it, we have to leave it in the nucleus while we copy it. This is like making a copy of a book from the Reserve shelf in the library. You are not allowed to leave with the book, so you have to make your copy before you leave the building. Now that we see our double strand of DNA, we want to have gene expression, to make a protein. The second task in protein synthesis (gene expression) is transcription (Takes place in the nucleus) First, unwind the gene by stepping apart from the second strand. One strand is the sense strand, used to code for a protein. We want to make a copy of that strand. Do I use this strand of my template? No. I use the other strand, the nonsense strand, called the antisense strand. If we used the sense strand, our protein would come out as a mirror image of what we want. When a black shirt (A) is on the antisense strand, it pairs with a white shirt (T), but we are going to use RNA to copy the antisense strand of DNA, and RNA does not have thymine (T), so it uses uracil (U) to bind to adenine (A), instead. I need some students with a brown shirt to represent uracil. Now, when I come across a black shirt on the antisense strand, I need a brown shirt. Continue to pair blue and grey as usual. When everyone on the antisense strand is paired up, the third strand needs to hold hands. Now we have made a copy of the antisense strand. The third strand we just made is called Messenger RNA (mRNA). We have just finished transcription. The mRNA must now leave the nucleus and head out toward the cytoplasm. Now that the mRNA has left, the two original strands of DNA come back together and bond. If someone lets go of a hand, the effect would be lethal. Ok, students in the DNA strand can sit down. The third task in protein synthesis (gene expression) is translation (Takes place in the cytoplasm) While the mRNA leaves the nucleus and enters the cytoplasm, it must stay in a line, and not bend or fold, or it will be attacked, degraded, and recycled for parts. Now we are in the cytoplasm with our single strand of mRNA, with Thymine replaced by uracil. A ribosome encompasses the first part of the strand. It reads the students in the strand, three at a time. Each set of three nucleotides (students) is a codon. Each codon is a code for a particular amino acid. The order of nucleotides of the first codon (first three students) codes for a particular amino acid, let’s say Tyrosine. This means that tyrosine will come and attach to the ribosome. The ribosome reads the next codon, which codes for the amino acid, histidine. That means that histidine will come and bind to the ribosome, next to tyrosine. Scientists have decoded the codon code…we know what amino acid is represented by each possible codon. The ribosome continues down the mRNA strand, reading three nucleotides at a time, and the amino acids line up in sequence, holding hands for dehydration synthesis. Before the ribosome is done reading the mRNA strand, another ribosome can come and start at the beginning of the same strand to start making a copy of the same mRNA strand. After all, you probably need more than one of the same protein. When the ribosome has finished reading the mRNA strand, and all of the amino acids will be present, lined up in proper order. This sequence of amino acids is the primary structure of the protein. If the AA’s bend into a staircase shape, that is secondary structure. If one AA holds the leg of another AA, that’s the tertiary structure. If there was another protein, and both proteins joined hands, that is the quaternary structure of a protein. That’s how a protein is made! Note that the mRNA left the nucleus as a single strand. That means that it was exposed, and other nucleotides in the cytoplasm could come and bind with it, inhibiting its ability to get to the ribosome to be read and make a protein. The mRNA could also pair with another mRNA strand, or a DNA strand. Scientists take advantage of the vulnerable mRNA strand to bind something onto it if the mRNA plans on making proteins as part of a tumor. In the future, we will be able to give a shot to someone with cancer, and it can cure them. It is already in clinical trials. It is not normal for uracil to be found in a DNA strand. If we see it there, it indicates a mutation has taken place. This occurs when cytosine undergoes a spontaneous chemical reaction and changes into uracil. This is an example of DNA damage. There are proteins in your nucleus whose job it is to scan DNA and replace appropriate nucleotides. Sun creates damage to the DNA strand by causing hydrogen bonds to cross link into covalent bonds. When this happens, the DNA strand cannot unwind to allow gene expression. Why are some people more prone to MI, HIV, cancer, diabetes? Gene expression. Certain receptors are expressed in the heart; when there is a mutation in that gene, they are more likely to have an MI (myocardial infarct, or heart attack) by age 50. You can have your DNA sequenced to see where your mutations are. Doctors are having hard time educating the public now; patients figure out what their problem might be, ask the doctor for medicine now, even though there is no problem yet. If the doctor gets their patent’s gene sequence, they don’t know what to do with it, need a molecular biologist to help them. Your mood changes your gene expression. Lonely, detached people get sick a lot. If attitude is everything, better pick a good one! How can you improve your mood? A simple touch from someone can elevate mood. For coffee lovers, just the smell of coffee wakes you up because it elevates your mood. Mood is improved by an increase in oxitocin production. Oxitocin does more than cause childbirth contractions and production of breast milk; men have oxytocin too, so it has other jobs, such as increasing your ability to bond socially. Infatuation causes oxytocin to increase. It is made in the hypothalamus, stored in the posterior pituitary (neurohypophysis), where you can also find ADH. To remember what these hormones do, ADH will make you swell, oxytocin you scream like hell. Men in orgasm are secreting high amounts of oxytocin. Mutation vs. DNA damage DNA damage is when the gene sequence of a cell is not in proper condition, but the daughter cells are normal. Mutation is an inheritable change in the gene sequence, so the daughter cells have the same damage. Stem cells can be pleuripotent or unipotent Who is determined to go into nursing? Pharmacy school? Physical Therapy? I can’t tell by looking at you what your determination is. With proper training in your graduate program, you will become a different person than you are now. Right now, you are determined. When you get your graduate degree, you will be differentiated. The ultimate stem cell is the one-cell zygote. It is pleuripotent, because it can turn into anything you direct it to be: blood, bone, muscle, nerve. Once it becomes determined to turn into a blood cell, it can no longer turn into a bone, muscle, or nerve cell. Now it is no longer pleuripotent. It is unipotent. Still, it is not fully differentiated yet, because, with further training, it can become a specific type of blood cell, like a white blood cell. Yet, it is still not fully differentiated, because it needs more training to become a specific type of white blood cell, say a B-cell. Differentiation is a process, from Pleuripotent to unipotent, to differentiated. When a cell is fully differentiated, it cannot turn into a T-cell or any other type of cell. Training makes you more and more special.