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AP Exam #5 Study Guide DNA History Scientists very unsure of what molecule contained genetic information. Initially thought it was proteins. Transforming Factor (Frederick Griffith). Trying to find a cure for pneumonia mixed harmless live bacteria with heat-killed infectious bacteria. Found that harmless became virulent. Called this transformation. Avery, McCarty & MacLeod wanted to know which molecule was the transforming factor. Injected protein into bacteria (No effect). Injected DNA into bacteria (Bacteria were transformed into virulent). Conclusion= DNA is transforming factor. Hershey and Chase- Confirmation of DNA as genetic material. Conducted blender experiment with bacteriophages. 2 separate media used 35S was used in their proteins, and 32P was used in their DNA. Infected bacteria with the labeled phages. Once the blender was used on the broth, they noticed that the radioactive proteins were in the supernatant (Not the pellet where the bacteria were). In the other group, they noticed that the radioactive material was in the pellet. They concluded that the 32P was transferred from the phages into the bacteria which were located in the pellet. Erwin Chargaff- Came up with the base pairing rules. A and T, C and G pair up in DNA. In humans: A= 30.9%, T=29.4%, G=19.9%, and C=19.8% Watson & Crick- Developed the double helix model. Structure of DNA DNA is a double helix (Two strands). It is looks like a spiral staircase. The two strands run anti-parallel or complimentary (mirror image). In DNA a purine (A or G) will always pair with a specific pyrimindine (T or C). A:T and C:G is the specific pairing. Held together with hydrogen bonds. Phosphodiester bonds= Phosphate to sugar bonds (The outsides of the ladder), are bonded at the 5’ and 3’ ends. Strands run in a 5’ to 3’ prime direction on one side of the ladder, and from 3’ to 5’ on the other side. Replication (Copying) of DNA Occurs during S phase of the cell cycle. Semi-conservative replication is the widely accepted model. 1 strand becomes 2 with each new DNA molecule receiving one original, and one new strand of DNA. Meselson & Stahl- labled nucleotides of parent DNA strands. Confirmed semiconservative replication theory. Replication occurs in a series of coordinated steps. Enzymes drive the process. Step 1- DNA is unwound with an enzyme called helicase. This causes a replication fork to form. The replication fork is stabilized with single-stranded binding proteins. There are multiple replication forks in a DNA molecule at one time. Step 2- New nucleotides are brought in to match up to the template strands. The new strand is built from 5’ to 3’. It is read from 3’ to 5’, but grows from 5’ to 3’. DNA polymerase III adds nucleotides, but it can only extend an existing molecule. In order to place the first base, a short RNA primer is built by an enzyme primase. Once primer is added, DNA polymerase III can start adding bases. Energy for all of this is provided by nucleosides. These nucleosides are converted to the nucleotides that are attached to the strand. Since DNA can only be read from 3’ to 5’, this creates an issue due to the complementary arrangement of the strands. The issue is that one strand replicates faster than the other creating a leading strand, and a lagging strand. The leading strand is replicated in a continuous manner. The lagging strand must be pieced together using Okazaki fragments, and a spot welding enzyme called ligase. Finally an enzyme called DNA polymerase I comes through and removes the RNA primers, and basically acts as a proofreader checking for any errors that may have occurred during replication. Every time that a copy of DNA is made, the DNA strand shortens by some bases (Telomeres) PCR (Polymerase Chain Reaction) allows scientists to make many copies of specific segments of DNA. Need just 1 molecule to make many copies. Making copies of DNA in a test tube. Items needed- Template DNA strand, DNA polymerase enzyme, nucleotides, and primer. Primers are crucial because they bracket the specific sequence you are looking for. Primers define the section to be cloned. Pretty simple process- take DNA and denature it with heat (900C), then cool it and build the DNA. Problem is that DNA polymerase is destroyed at (90 0C). Have to find another molecule that does the same thing, but can withstand heat (Taq Polymerase). Taq polymerase is found in hot springs bacteria. Transcription Transcribed DNA strand= template strand. Un-transcribed DNA strand= coding strand. Transcription bubble is formed. RNA polymerase in charge of transcription. Step 1 Initiation- RNA polymerase binds to a promoter sequence. Promoter is a starting point, tells which strand to read, and the direction in which it is read. DNA is always read from 3’ to 5’. Step 2 Elongation- RNA polymerase unwinds DNA ~20 base pairs at a time. There is no proofreading going on because many RNA copies are made, and there is a short shelf life. Step 3 Termination- RNA polymerase stops at the termination sequence. mRNA then leaves the nucleus through the nuclear pores. 3 RNA polymerase enzymes. RNA poly 1- only transcribes rRNA, RNA poly II- only transcribes genes into mRNA, and RNA poly III- only transcribes rRNA genes. Once mRNA is created, there needs to be some additional work to the mRNA strand. Cell must protect mRNA from enzymes that might break it down. It does this by adding a 5’ G cap, and a poly A tail to the 3’ end. In eukaryotic cells, there is a lot of junk in the strand of RNA. This non-coding junk is called an intron. Introns must be cut out, so that an RNA strand that is information only can be formed. Information is called an exon. Introns are cut out by molecules called spliceosomes. Introns are cut out, and then the exons are pasted back together. RNA can also act as an enzyme (ribozyme) Remember splicing has to be exactly accurate. No mistakes can be made in the changing of DNA to RNA. Frameshift Mutation- Occurs when there is an addition or deletion. Remember the frame is read in three letter chunks called codons. One additional, or one less throws the frame off and leads to the complete protein not being made. Point Mutation- Happens at a point in the DNA strand where one or a few letters get changed. Not as bad due to having the same number of letters. Protein will still get made, it may be different from original. Translation Codons- blocks of 3 nucleotides that “code” for an amino acid. This is the strongest support for a common ancestor. (All organism have a common code). Code is redundant- several codons code for each amino acid. (This prevents point mutations from being overly harmful) Start Codon- AUG (School starts in August). Ribosomes need an “on” switch to initiate translation. Stop Codons- UAA, UAG, UGA. Off switch for ribosomes. mRNA- strand that gets read rRNA- RNA that is in charge of reading mRNA tRNA- RNA that is in charge of bringing the amino acid. tRNA is shaped like a clover leaf. Amino acids attach at the 3’ end of the clover. tRNA must have amino acid loaded onto it. This is done using the enzyme Aminoacyl tRNA synthase. It is an endergonic reaction that is unstable. It needs to be unstable so that the amino acid can easily be removed from tRNA. Ribosomes- Site where translation occurs. It is made up of rRNA and proteins. It is composed of 2 subunits a large and a small. There are three sites inside the ribosome that are used for building the protein. E, P, and A. P Site- where the polypeptide chain grows. Amino acid is added to the chain A Site- holds the tRNA that is next in line to drop off amino acid (On deck circle) E Site- site where empty tRNA is released back into the cell. Translation happens in three steps. Initiation, Elongation, and Termination Initiation- mRNA is brought to ribosome and begins to slide through (Dollar bill in vending machine) Elongation- As mRNA is being read, the polypeptide chain grows. Termination- Release factor is used (Protein) to bond to A site. Usually a water molecule to polypeptide chain. Protein is made and translation is done. What happens to protein once made?? It is used for any number of things depending on the protein. Secretion, mitochondria may need it, golgi apparatus might take it and modify it, could be a used structurally. Viruses Basically a package of genes in transit from one host cell to another. A virus is considered to be emerging if it jumps host species. Ex- Ebola, SARS, bird flu, hantavirus. A virus is composed of DNA or RNA enclosed in a protein coat. Viruses are not cells. They are extremely tiny needing an electron microscope to be seen. Viruses lack enzymes for metabolism, ribosomes for protein synthesis, and need host machinery to replicate and function. Viral genomes can vary significantly depending on the family of virus one belongs to. You can have double-stranded or single-stranded DNA. There are viruses that have double or single-stranded RNA. DNA can be linear or circular depending on the virus. Protein coat for viruses is called a capsid. It is a crystal like shell. Some viruses have another outer covering called a viral envelope. It is a lipid bilayer that is used to hide the virus. Viral envelope is used to provide camouflage to virus in order to help it attack. Two different lifecycles: Lytic and Lysogenic Lytic- reproduce in host cell, and then spread to other parts of body by causing cell lysis (breaking). Lysogenic- integrates DNA into host and reproduces with cell. Most of the time, will enter a lytic stage at some point. RNA viruses- Called retroviruses. Contain an enzyme called reverse transcriptase. Can change RNA into DNA. By doing this, the cell now produces viral mRNA. This in turn causes host to produce viral proteins. Biotechnology DNA sequencing developed by Fred Sanger. Idea is to synthesize DNA in vitro. Creates sequences to study genes. Human Genome Project- US government project. Designed to map out the entire human genome. Arms race established between government group, and private group. All genes for humans are mapped and available online free of charge. There are roughly 30,000 human genes. Transposons- a piece of DNA that can move from one location to another. These genes allow for mutations to occur within an organism’s genome. Gene manipulation is not a new science per say. We have been controlling outcomes in plants and animals for years. Artificial selection- choosing traits we like best, and breeding those traits in organisms. New science is found in genetic engineering. Instead of breeding, we manipulate the DNA directly. In order to manipulate genes, we need a set of tools that we can use. Basic idea is, we can cut DNA up (restriction enzymes), then paste it (ligase). Once we have the template we want, we can copy it using plasmids, and finally we can find the gene we are looking for through PCR (Creating multiple copies). This stuff is used when we are talking about finding cures for diseases, producing certain proteins that may benefit our population, and any other private uses that may be necessary.