Study Guide

Gene to Protein In the Real World

  • Health

    "Let's talk about sex, baby; Let's talk about you and me; Let's talk about all the good things; and the bad things that may be." That's right Salt-n-Pepa, it is time to talk about sex….sex chromosomes, actually. It may not have been what Salt-n-Pepa was talking about but we will talk about the bad things that could be when it comes to sex determination.

    Male humans have an X and a Y chromosome, while females have two X chromosomes. The X and Y chromosome actually have few of the same genes, which means that a male has only one copy of genes on the X. Females instead have two copies. In a female one X comes from the father and one X is provided from the mother. The more the merrier? Not exactly.

    Therefore, the cell needs to find a way to silence (prevent expression of) one X chromosome in a XX individual. The cells in your body actually do this in random fashion early in development. Therefore, the X chromosomes from dad may be inactivated in one cell, while the X chromosome that comes from mom may be inactivated in another cell. This is called genetic mosaicism.


    You can actually visualize genetic mosaics in some animals where the gene for coat color occurs on the X chromosome. If the father and mother of an animal provide genes for different coat colors, then the coat color will be a patchwork. Calico cats are an excellent example. Since the gene is present on the X chromosome, calico cats are predominantly female.

    A calico cat has a gene for orange coat color on one X chromosome and genes for black or white coat color on the other X chromosome. Since females inactivate one X chromosome at random, the calico cat's coat will be a mix of the colors. Some cells will express the coat color from one x chromosome, while others will express the coat color from the other x chromosome.

    Can you imagine what would happen if the genes for human pigmentation where on the X chromosome? How would it affect the appearance of a female human? A male human?

    In some special instances, a person can get more than two X chromosomes. A person who is XXY is male has Klinefelter's syndrome (a condition of having an extra X). Alternatively, a person who has one X is female, a condition called Turner's syndrome. What this fact tells us is that it is the presence of the Y that determines sex in humans. However, it is not that straightforward. Turner's syndrome patients are infertile, and have a number of other health concerns. The same can be said for Klinefelter's syndrome.

    The process of X silencing is called X inactivation. X inactivation results in the heterochromatization of the chromosome. If you look at a female's cells under the microscope you can actually see the chromosome that has been inactivated. It is a clump in the nucleus—a structure called a Barr body. This chromosome won't be transcribed.

    You may be wondering how this process of inactivation works. You've learned from the regulation theme that there are lots of ways to interfere with the production of a protein. We've told you that X inactivation is transcriptional inactivation. This process occurs through the use of a special RNA, called Xist that actually promotes chromatin changes in the structure of the X chromosome. This makes it take on a more condensed structure that is less accessible for the transcription machinery.

    Red Alert. It turns out that x inactivation doesn't always work by silencing one chromosome. If you look throughout different organisms you will see the many different solutions that organisms have arrived at to limit the amount of transcription from the X sex chromosome. Some dial down transcription down by half as much on one chromosome. The way that the cell does this is called dosage compensation.

  • Biotechnology

    Genetic Engineering

    Humans have been messing with genes throughout human history. We've used genetics to make better animals for consumption, to make plants more resistant to disease, and to influence the genetics of our children by selecting for specific traits in a mate. But in this section, we will talk about a different type of "messing" with genes, a process called biotechnology, or genetic engineering.

    Genetic engineering refers to the field where scientists can directly manipulate the production of a protein or RNA for human products. In this section we will go over some of the methods scientists use. It is kind of like building with Legos…

    First, let's talk about cloning. In the media you will probably hear about cloning more often in the terms of creating a whole animal (remember Dolly the sheep, anyone?) or the possibility of cloning a human being. Rather than talking about cloning an organism, we will talk about a type of cloning that is much less controversial and much more common: the cloning of a gene. In fact, many of the products you use on a daily basis are created with the help of this type of molecular cloning.

    It is common in the field of biotechnology to isolate a gene that encodes for a particularly useful protein or RNA product. Scientists can create more of this DNA, and even modify the gene such that the protein product is better suited for a scientist's needs. But, first thing's first. Let's talk about the steps of cloning a DNA fragment.

    1. First the fragment containing the gene of interest must be isolated, or cut out of a mass of DNA.
        
    2. The piece of DNA must be ligated, or glued into another DNA molecule. For example, the gene can be transferred to make a circular piece of DNA called a plasmid.
        
    3. This piece of DNA is then transferred into a cell through a process called transfection.
        
    4. Scientists then select for cells that contain the cloned DNA. They can use the cells to express large amounts of the gene interest.

    Many gene products are produced this way, often growing the product in bacterial cells or simple eukaryotic cells such as yeast. One of the first applications of this technology was insulin. Diabetics today get their insulin from genetically modified bacteria containing the insulin gene. Vaccines are also often produced with the help of genetic engineering.

    In the laboratory, scientists commonly use genetic engineering to make genetically modified organisms. Now we know you might be thinking of Frankenstein here, but in reality these organisms are a lot less scary (but much cooler). Let's talk a little bit about the genetic engineer's bag of tricks.

    Scientists often make gene knockouts, where one or more of an organisms genes is made to not function. The initial steps to making a gene knockout are pretty much the same as cloning a gene.

    1. The scientists make a DNA construct that is targeted to the gene locus.
        
    2. The construct can be put into a cell, specifically embryonic cells. These embryonic cells are put into an early embryo.
        
    3. The DNA is taken up by the embryo, a term called recombination, and sometimes incorporated into the germ line.
        
    4. Resulting generations can be bred that do not contain the gene of interest.
        
    5. Animals can then be studied to understand the function of the gene that was eliminated.

    Other times scientists create knock-in animals. In this case, a new gene is inserted into the genome of an organism. There are all sorts of uses of gene knock-ins. One example is that a fluorescent protein can be inserted under the control of a specific promoter. Whenever the promoter orders gene expression, the knocked in gene will be expressed.

    Alternatively, a fluorescent protein, or tag, can also be added on to a gene that a scientist is trying to study. The result is that the protein will fluoresce in the animal. Scientists will be able to tell exactly where a particular protein is, or if it is specially localized to specific cells or tissues. This technique has been used in fish to "light up" regions of the nervous and digestive systems. Fluorescent kittens have been used for AIDS research. In many ways, genetic engineering is only limited by the imagination of scientist.

    Some human disorders can be linked to a mutation in a specific gene. Some examples are cystic fibrosis, hemophilia, and sickle cell anemia. The ability to replace a "faulty" gene with a "good" copy in humans would be a huge advantage. Gene therapy is the way that scientists attempt to do this feat. Scientists have had some success at using viruses and liposomes to introduce genes into a host's genome, although this area of research is still in early stages.

    Genetic engineering also offers the opportunity for the creation of organisms that benefit humans. Our ability to understand human diseases has been extremely advanced by studying genetically engineered animals. However, the tools can also be used for other purposes.

    Allerca has reportedly engineered hypoallergenic dogs and cats. According to Allerca, you can now snuggle with one of their furry felines without red eyes and itching….for about $7000 that is. The company has been somewhat controversial, but it none-the-less spotlights how genetic engineering has the potential to drastically change the way we live.

    The Simpsons gene, according to the TV show, is the mutated gene that contributes to Homer's baldness and laziness. It is only present on the Y chromosome, which explains why only the men in the Simpson family have the trait. Sound like pure TV ridiculousness?

    Scientists have found that deletion of a gene in mice, called RGS14, actually makes mice smarter. Perhaps there is a Homer Simpson gene after all. Can you envision the possibilities? No, we are not talking about making genetically engineered kids here. But, what if you could find a drug that targeted the same gene? Genetic engineering brings up some sticky questions and a lot of murky answers.

    Getting rid of a gene should never be taken lightly. As you have learned, genes play some important functions. It is not so straightforward to get rid of a gene and have everything be okay. Sometimes getting rid of a gene can have some severe consequences, even resulting in the death of the organism. Therefore, scientists need to be ethical and responsible. Don't believe us? Watch I Am Legend. In the movie genetic engineering results in the production of a virus that is supposed to cure cancer. Instead the virus mutates and spreads throughout the world, killing most of the world's population. Of course, things are often a bit more dramatic in Hollywood….

  • Research

    In 1998, a group of scientists injected double stranded RNA into the worm Caenorhabditis elegans. This introduction of the RNA into the worm was able to completely silence the corresponding gene such that no protein was produced. But, who cares about a worm, right?

    Wrong. This set of research experiments introduced an entirely novel mechanism of gene regulation that occurs even in humans. These observations, and the work that followed it, would generate a tool used widely in research labs throughout the world.

    Eight years later the Nobel Prize was awarded to 2 of these researchers: Craig Mello and Andy Fire. Their finding, initially discovered in the worm, offers an opportunity to understand proteins and their functions, and a potential therapeutic tool to cure disease. Who knew that a little underdog worm could become a science all star?

    Before 1998, there had been a lot of confusing observations that RNA could silence a gene. It made some sense that an antisense transcript could silence an mRNA, potentially by binding to the transcript and preventing its translation. The confusing observation was that both the sense and antisense strands to the mRNA were effective. Furthermore, the silencing often lasted more than one generation, even though most RNAs were degraded.

    Let's talk a little bit about the experiment in 1998. The scientists studied the gene unc-22, whose protein product is important for worm mobility. Inactivation of the unc-22 gene results in a worm that twitches, or is completely unable to move. The authors showed that the injection of double stranded RNA was the most effective at silencing the unc-22 gene, resulting in twitching or loss of mobility. The ds-RNA had to be complementary to the unc-22 gene for the silencing to be effective. Similar scenarios where observed when double stranded RNA was injected to silence different genes. This process was coined RNAi, or RNA interference.

    This landmark study raised more questions than it answered. It was still unclear whether the gene was silenced at the level of transcription or translation. Nonetheless, this study provided some important observations. It provided a mechanism by which genes could be silenced at will. The worm was an ideal organism for these studies because of the ease of injections. Later work showed that worms could also be fed the RNAs or simply soaked in them. Up until recently this technique was thought to be limited to worms. It turns out though that we may be able to eat food and influence the gene expression of genes in our bodies. Interested? Check out this brain snack.

    Brain Snack

    Check out this awesome link about how RNA from rice can influence gene expression. http://blogs.discovermagazine.com/80beats/2011/09/21/what-you-eat-affects-your-genes-rna-from-rice-can-survive-digestion-and-alter-gene-expression/

    While much remains unknown about the details of RNAi, we do know have some more insights into the process. It is a natural defense mechanism against viruses. We know that double stranded RNAs are processed into small interfering RNAs by an endonuclease called Dicer. This then interferes with translation.

    In addition to the direct silencing of translation, more recently researchers showed that some of the components of the RNAi machine are used to affect gene regulation pre transcription. Here small RNAs can actually alter the expression of a gene by changing the chromatin environment surrounding a gene. By making a gene more heterochromatic, the genes are less open and able to recruit the necessary transcription factors.

    We mentioned that RNAi is a powerful tool to study disease states. What do we mean? Many human conditions result from the loss of expression of a gene product. Now imagine that a researcher can design an experiment that lets him effectively "turn off" that gene in an otherwise healthy cell. The researcher can begin to ask questions about why the disease state occurs at the cellular level.

    Other times disease states are thought to occur because of the overproduction of a gene product. In this case researchers and drug companies are figuring out ways to selectively introduce small RNAs to humans in order to combat the overproduction of the gene product. The delivery of the small RNAs to the correct cells in the human body is one of the most significant challenges of RNAi therapeutics. This same strategy can be used to target cancer cells. Can you think of how might RNAi be used to induce the death of a cancer cell?