Practice problems for these concepts can be found at: Molecular Genetics Review Questions for AP Biology
Gene Expression
Let's cover some vocabulary before diving into this section:
Promoter region: a base sequence that signals the start site for gene transcription; this is where RNA polymerase binds to begin the process.
Operator: a short sequence near the promoter that assists in transcription by interacting with regulatory proteins (transcription factors).
Operon: a promoter/operator pair that services multiple genes; the lac operon is a wellknown example (Figure 11.7).
Repressor: protein that prevents the binding of RNA polymerase to the promoter site.
Enhancer: DNA region, also known as a "regulator," that is located thousands of bases away from the promoter; it influences transcription by interacting with specific transcription factors.
Inducer a molecule that binds to and inactivates a repressor (e.g., lactose for the lac operon).
The control of gene expression is vital to the proper and efficient functioning of an organism. In bacteria, operons are a major method of gene expression control. The lactose operon services a series of three genes involved in the process of lactose metabolism. This contains the genes that help the bacteria digest lactose. It makes sense for bacteria to produce these genes only if lactose is present. Otherwise, why waste the energy on unneeded enzymes? This is where operons come into play—in the absence of lactose, a repressor binds to the promoter region and prevents transcription from occurring. When lactose is present, there is a binding site on the repressor where lactose attaches, causing the repressor to let go of the promoter region. RNA polymerase is then free to bind to that site and initiate transcription of the genes. When the lactose is gone, the repressor again becomes free to bind to the promoter, halting the process.
Because gene expression in eukaryotes involves more steps, there are more places where gene control can occur. Here are a few examples of eukaryotic gene expression control:
Transcription: controlled by the presence or absence of particular transcription factors, which bind to the DNA and affect the rate of transcription.
Translation: controlled by factors that tend to prevent protein synthesis from starting. This can occur if proteins bind to mRNA and prevent the ribosomes from attaching, or if the initiation factors vital to protein synthesis are inactivated.
DNA methylation: addition of CH3 groups to the bases of DNA. Methylation renders DNA inactive. Barr bodies, discussed in Chapter 10, are highly methylated.
These are only a few of the examples of gene expression control that occur in eukaryotes. Do not get lost in the specifics.
The Genetics of Viruses
A virus is a parasitic infectious agent that is unable to survive outside of a host organism. Viruses do not contain enzymes for metabolism, and they do not contain ribosomes for protein synthesis. They are completely dependent on their host. Once a virus infects a cell, it takes over the cell's machinery and uses it to produce whatever it needs to survive and reproduce. How a virus acts after it enters a cell depends on what type of virus it is. Classification of viruses is based on many factors:
Genetic material: DNA, RNA, protein, etc.?
Capsid: type of capsid?
Viral envelope: present or absent?
Host range: what type of cells does it affect?
All viruses have a genome (DNA or RNA), and a protein coat (capsid). A capsid is a protein shell that surrounds the genetic material. Some viruses are surrounded by a structure called a viral envelope, which not only protects the virus but also helps the virus attach to the cells that it prefers to infect. The viral envelope is produced in the endoplasmic reticulum (ER) of the infected cell and contains some elements from the host cell and some from the virus. Each virus has a host range, which is the range of cells that the virus is able to infect. For example, the HIV virus infects the T cells of our body, and bacteriophages infect only bacteria.
A special type of virus that merits discussion is one called a retrovirus. This is an RNA virus that carries an enzyme called reverse transcriptase. Once in the cytoplasm of the cell, the RNA virus uses this enzyme and "reverse transcribes" its genetic information from RNA into DNA, which then enters the nucleus of the cell. In the nucleus, the newly transcribed DNA incorporates into the host DNA and is transcribed into RNA when the host cell undergoes normal transcription. The mRNA produced from this process gives rise to new retrovirus offspring, which can then leave the cell in a lytic pathway. A well-known example of a retrovirus is the HIV virus of AIDS.
Once inside the cell, a DNA virus can take one of two pathways—a lytic or a lysogenic pathway. In a lytic cycle, the cell actually produces many viral offspring, which are released from the cell—killing the host cell in the process. In a lysogenic cycle, the virus falls dormant and incorporates its DNA into the host DNA as an entity called a provirus. The viral DNA is quietly reproduced by the cell every time the cell reproduces itself, and this allows the virus to stay alive from generation to generation without killing the host cell. Viruses in the lysogenic cycle can sometimes separate out from the host DNA and enter the lytic cycle. (Like a bear awaking from hibernation.)
Viruses come in many shapes and sizes. Although many viruses are large, viroids are plant viruses that are only a few hundred nucleotides in length, showing that size is not the only factor in viral success. Another type of virus you should be familiar with is a prion—an incorrectly folded form of a brain cell protein that works its magic by converting other normal host proteins into misshapen proteins. An example of a prion disease that has been getting plenty of press coverage is "mad cow" disease. Prion diseases are degenerative diseases that tend to cause brain dysfunction—dementia, muscular control problems, and loss of balance.
The Genetics of Bacteria
Bacteria are prokaryotic cells that consist of one double-stranded circular DNA molecule. Present in the cells of many bacteria are extra circles of DNA called plasmids, which contain just a few genes and have been useful in genetic engineering. Plasmids replicate independently of the main chromosome. Bacterial cells reproduce in an asexual fashion, undergoing binary fission. Quite simply, the cell replicates its DNA and then physically pinches in half, producing a daughter cell that is identical to the parent cell. From this description of binary fission, it seems unlikely that there could be variation among bacterial cells. This is not the case, thanks to mutation and genetic recombination. As in humans, DNA mutation in bacteria occurs very rarely, but some bacteria replicate so quickly that these mutations can have a pronounced effect on their variability.
Transformation
An experiment performed by Griffith in 1928 provides a fantastic example of transformation— the uptake of foreign DNA from the surrounding environment. Transformation occurs through the use of proteins on the surface of cells that snag pieces of DNA from around the cell that are from closely related species. This particular experiment involved a bacteria known as Streptococcus pneumoniae, which existed as either a rough strain (R), which is nonvirulent, or as a smooth strain (S), which is virulent. A virulent strain is one that can lead to contraction of an illness. The experimenters exposed mice to different forms of the bacteria. Mice given live S bacteria died. Mice given live R bacteria survived. Mice given heatkilled S bacteria survived. Mice given heat-killed S bacteria combined with live R bacteria died. This was the kicker … all the other results to this point were expected. Those exposed to heat-killed S combined with live R bacteria contracted the disease because the live R bacteria underwent transformation. Some of the R bacteria picked up the portion of the heatkilled S bacteria's DNA, which contained the instructions on how to make the vital component necessary for successful disease transmission. These R bacteria became virulent.
Transduction
To understand transduction, you first need to be introduced to something called a phage (Figure 11.8)—a virus that infects bacteria. The mechanism by which a phage virus infects a cell reminds me of a syringe. A phage contains within its capsid the DNA that it is attempting to deliver. A phage latches onto the surface of a cell and like a syringe, fires its DNA through the membrane and into the cell. Transduction is the movement of genes from one cell to another by phages. The two main forms of transduction you should be familiar with are generalized and specialized transduction.
Generalized Transduction Imagine that a phage virus infects and takes over a bacterial cell that contains a functional gene for resistance to penicillin. Occasionally during the creation of new phage viruses, pieces of host DNA instead of viral DNA are accidentally put into a phage. When the cell breaks, expelling the newly formed viral particles, the phage containing the host DNA may latch onto another cell, injecting the host DNA from one cell into another bacterial cell. If the phage attaches to a cell that contains a nonfunctional gene for resistance to penicillin, the effects of this transduction process can be observed. After injecting the host DNA containing the functional penicillin resistance gene, crossover could occur between the comparable gene regions, switching the nonfunctional gene with the functional gene. This would create a new cell that is resistant to penicillin.
Specialized Transduction This type of transduction involves a virus that is in the lysogenic cycle, resting quietly along with the other DNA of the host cell. Occasionally when a lysogenic virus switches cycles and becomes lytic, it may bring with it a piece of the host DNA as it pulls out of the host chromosome. Imagine that the host DNA it brought with it contains a functional gene for resistance to penicillin. This virus, now in the lytic cycle, will produce numerous copies of new viral offspring that contain this resistance gene from the host cell. If the new phage offspring attaches to a cell that is not penicillin resistant and injects its DNA and crossover occurs, specialized transduction will have occurred.
Conjugation
This is the raciest of the genetic recombinations that we will cover … the bacterial version of sex. It is the transfer of DNA between two bacterial cells connected by appendages called sex pili. Movement of DNA between two cells occurs across a cytoplasmic connection between the two cells and requires the presence of an F plasmid, which contains the genes necessary for the production of a sex pilus.
Genetic Engineering
DNA technology is advancing at a rapid rate, and you need to have a basic understanding of the most common laboratory techniques for the AP Biology exam.
Restriction enzymes are enzymes that cut DNA at specific nucleotide sequences. When added to a solution containing DNA, the enzymes cut the DNA wherever the enzyme's particular sequence appears. This creates DNA fragments with single-stranded ends called "sticky-ends," which find and reconnect with other DNA fragments containing the same ends (with the assistance of DNA ligase). Sticky ends allow DNA pieces from different sources to be connected, creating recombinant DNA. Another concept important to genetic engineering is the vector, which moves DNA from one source to another. Plasmids can be removed from bacterial cells and used as vectors by cutting the DNA of interest and the DNA of the plasmid with the same restriction enzyme to create DNA with similar sticky ends. The DNA can be attached to the plasmid, creating a vector that can be used to transport DNA.
Gel Electrophoresis
This technique is used to separate and examine DNA fragments. The DNA is cut with our new friends, the restriction enzymes, and then separated by electrophoresis. The pieces of DNA are separated on the basis of size with the help of an electric charge. DNA is added to the wells at the negative end of the gel. When the electric current is turned on, the migration begins. Smaller pieces travel farther along the gel, and larger pieces do not travel as far. The bigger you are, the harder it is to move. This technique can be used to sequence DNA and determine the order in which the nucleotides appear. It can be used in a procedure known as Southern blotting (after Edwin M. Southern, a British biologist) to determine if a particular sequence of nucleotides is present in a sample of DNA. Electrophoresis is used in forensics to match DNA found at the crime scene with DNA of suspects. This requires the use of pieces of DNA called restriction fragment length polymorphisms (RFLPs). DNA is specific to each individual, and when it is mixed with restriction enzymes, different combinations of RFLPs will be obtained from person to person. Electrophoresis separates DNA samples from the suspect and whatever sample is found at the scene of the crime. The two are compared, and if the RFLPs match, there is a high degree of certainty that the DNA sample came from the suspect. In Figure 11.9, if well A is the DNA from the crime scene, then well C is the DNA of the guilty party.
Steve (12th grade): "Know this cold. It was all over my exam!"
Cloning
Sometimes it is desirable to obtain large quantities of a gene of interest, such as insulin for the treatment of diabetes. The process of cloning involves many of the steps we just mentioned. Plasmids used for cloning often contain two important genes—one that provides resistance to an antibiotic, and one that gives the bacteria the ability to metabolize some sugar. In this case, we will use a galactose hydrolyzing gene and a gene for ampicillin resistance. The plasmid and DNA of interest are both cut with the same restriction enzyme. The restriction site for this enzyme is right in the middle of the galactose gene of the plasmid. When the sticky ends are created, the DNA of interest and the plasmid molecules are mixed and join together. Not every combination made here is what the scientist is looking for. The recombinant plasmids produced are transformed into bacterial cells. This is where the two specific genes for the plasmid come into play. The transformed cells are allowed to reproduce and are placed on a medium containing ampicillin. Cells that have taken in the ampicillin resistance gene will survive, while those that have not will perish. The medium also contains a special sugar that is broken down by the galactose enzyme present in the vector to form a colored product. The cells containing the gene of interest will remain white since the galactose gene has been interrupted and rendered nonfunctional. This allows the experimenter to isolate cells that contain the desired product. Now, it is time for us to quit cloning around and move onto another genetic engineering technique.
Polymerase Chain Reaction
Think of this technique as a high-speed copy machine. It is used to produce large quantities of a particular sequence of DNA in a very short amount of time. If the cloning reaction is the 747 of copying DNA, then polymerase chain reaction (PCR) is the Concorde. This process begins with double-stranded DNA containing the gene of interest. DNA polymerase, the superstar enzyme of DNA replication, is added to the mixture along with a huge number of nucleotides and primers specific for the sequence of interest, which help initiate the synthesis of DNA. PCR begins by heating the DNA to split the strands, followed by the cooling of the strands to allow the primers to bind to the sequence of interest. DNA polymerase then steps up to the plate and produces the rest of the DNA molecule by adding the nucleotides to the growing DNA strand. Each cycle concludes having doubled the amount of DNA present at the beginning of the cycle. The cycle is repeated over and over, every few minutes, until a huge amount of DNA has been created. PCR is used in many ways, such as to detect the presence of viruses like HIV in cells, diagnose genetic disorders, and amplify trace amounts of DNA found at crime scenes.
Practice problems for these concepts can be found at: Molecular Genetics Review Questions for AP Biology
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