Genetics Exam 3 Study Guide
Genetics Exam 3 Study Guide 85033 - GEN 3000 - 002
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85033 - GEN 3000 - 002
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This 30 page Study Guide was uploaded by Toni Franken on Friday March 4, 2016. The Study Guide belongs to 85033 - GEN 3000 - 002 at Clemson University taught by Kate Leanne Willingha Tsai in Summer 2015. Since its upload, it has received 271 views. For similar materials see Fundamental Genetics in Biomedical Sciences at Clemson University.
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GEN 3000 – Exam 2 2016 Study Guide Chapters 9 and 11 Dr. Tsai, Clemson University Exam 2: Chapters 9 and 11 Modeled around Dr. Tsai’s recommended subjects to study – color coded/organized by chapter. Chapter 9: DNA Structure and Analysis Four qualifications of Genetic Material: o Must be capable of being replicated faithfully o Must encode and store complex information o Must contain enough information to yield phenotype o Must have variation as a result of mutation Transforming principle: The hypothesized factor involved in the transformation of genetic material o Griffith: Used Streptococcus pneumonia – found that the virulent strain (S) had a smooth, polysaccharide coat. The antivirulent strain (R) lacked a coat entirely. When heat killed S strain was combined with a viable R strain, some R could spontaneously S. Griffith concluded that some type of interaction had taken place between the viable R strain and the heat killed S strain to allow for virulent S to be produced. He called this unknown material the Transforming Principle. o Avery, MacLeod, McCarty: In 1944, these three scientists isolated what they believed to be the transforming principle, and what they believed to be DNA. They used both proteases (protein destroying enzymes) and RNases (RNA destroying enzymes) in cultures of the R and heatkilled S strains of S. pneumonia. They found that this did not stop the transforming principle from working, and virulent strains of S were still produced. However, when they used DNase (DNA destroying enzymes), the virulence of S was never achieved. This led them to conclude that DNA was the transforming principle. o Hershey and Chase: In 1952, these men used a T2 Bacteriophage (also simply called a phage) to build a case for nucleic acid as genetic material. Remember, viruses can hijack the genetic material and replication mechanisms of their host bacteria. They will inject their own genetic material into the host cell. Hershey and Chase took advantage of this. In one group of bacteriophages, they marked proteins with a radioactive sulfur compound, and then the infected bacteria were allowed to replicate. The progeny had no radioactively labeled proteins transferred. In another group of bacteriophages, they marked nucleic acids with a radioactive phosphorous. The infected, unmarked bacteria were allowed to replicate, and the progeny readily displayed the radioactive material, suggesting that nucleic acids, either RNA or DNA, were the genetic material of cells. NOTE: They were able to separate the viruses and bacteriophages by centrifuging the mixture and putting it through a blender. This separated the viruses and cells, and stripped the coats, leaving only the compounds they wished to observe. Nucleic acids: o Tobacco Mosaic Virus: Displays the concept that RNA can act as genetic material. Has a single piece of RNA surrounded by protein, a very simple design. We can show recombination of the RNA by washing away the protein, and combining strains A and B, getting hybrids. o Purine vs. pyrimidine: (structures will not be tested) Purine: Adenine and Guanine are the purine bases of DNA, and they complement the pyrimidine bases. These are larger than pyrimidines. Pyrimidine: Cytosine, Thymine (DNA), and Uracil (RNA) are the pyrimidine bases of genetic material. Matches: DNA: A/T:G/C RNA: A/T:G/U Nucleotide components: o Sugar: Deoxyribose in DNA, and Ribose in RNA. These are attached to a nitrogen containing base. o Base: Nitrogen containing bases consist of purines and pyrimidines. See above for different variations. NOTE: The sugar attached to its respective base is called a NUCLEOSIDE, whereas a NUCLEOTIDE is a nucleoside attached to a phosphate group. o Phosphate Backbone: The backbone of DNA is made up of phosphate groups, which are attached to the nucleosides. o Types: Deoxyadenosine 5’ monophosphate (Deoxyribose sugar, adenine nitrogen base, attached to a phosphate) dAMP Deoxyguanosine 5’ monophosphate (Deoxyribose sugar, guanine nitrogen base, attached to a phosphate) dGMP Deoxycitidine 5’ monophosphate (Deoxyribose sugar, cytosine nitrogen base, attached to a phosphate) dCMP Deoxythymidine 5’ monophosphate (Deoxyribose sugar, thymine nitrogen base, attached to a phosphate). dTMP Chargaff’s rule: o Chargaff and his colleagues were able to separate the residues of the bases of nucleic acids, and found several things: The amount of Guanine residue was equal to that of the Cytosine residue, suggesting that this pyrimidine and purine match up in DNA. The amount of Thymine residue was equal to that of Adenine residue, suggesting that this pyrimidine and purine match up in DNA. Due to proportionality, the amount of (A+C) = (T+G). Watson, Crick (Wilkins, Franklin): o Wilkins and Franklin – Used xray crystallography to identify the location of molecules contained in DNA. This truly was the key to unlocking the secrets of DNA structure. o Watson and Crick: 1953 – Watson and Crick used chemistry and Xray diffraction to solve DNA structure (Franklin’s data was key!) “We have also been stimulated by a knowledge of the general nature of the unpublished experimental results and ideas of Dr. M. H. F. Wilkins, Dr. R. E. Franklin….” Proposed a double helical form of DNA made of matched pyrimidine and purine bases. If it were made of just matched purines, the structure would be too thick. If it were made of just matched pyrimidines, the structure would be too thin. The complementary bases instead matched the models put together by W and C. DNA characteristics o Bonds: Hydrogen bonds: Between the nitrogencontaining bases (pyrimidines and purines), hydrogen bonds are formed. There are three H bonds between G and C, and two H bonds between A and T. The fewer H bonds, the weaker the bond is, and the more easily it is broken. These H bonds are hydrophobic. These weaker bonds allow for the opening of DNA for replication/translation/transcription. Phosphodiester Bonds: The sugarphosphate backbone of DNA is connected by phosphodiester bonds, which are incredibly strong and hydrophilic. This keeps the DNA shape, and is very hard to break. o Directionality: The helical formation of DNA is made of two complementary strands. These strands run antiparallel to each other. The 5’ end of one (ending with a phosphate) matches to the complementary 3’ end (ending with a sugar) of the other strand. The nitrogen containing bases are in between these strands, and are connected by hydrogen bonds. NOTE: DNA can only replicate 5’ to 3’!!!! This is very important!!!! Will start at the 3’ end of the strand so that the 5’ end of the new strand can begin. This alternating complementary strand winds in a righthanded or clockwise spiral, also called an alpha helix. There are about 10 base pairs per rotation of the DNA strand. There is a major groove (larger) and minor groove (smaller) that is formed in the helical shape of DNA. o These characteristics are all relevant to BDNA (Beta DNA). This is the DNA type that Watson and Crick discovered, and it is the most stable form, which is present around water. But there are other types of DNA, also. A Form: The A form is a squished down version of the B form of DNA. This has been found outside of the cell. Outside of the cell, the DNA structure can modify. All of the antiparallel, complimentary is still true, just compacted, making the DNA smaller. The Z Form: This DNA form has a zigzag backbone, and has a left handed helix instead of a righthanded. Makes it stretched out and thin, and has been found in the cell. It’s not that the entire strand will suddenly switch, but regions of it will switch over. Found a correlation between active genes and ZDNA. If you were trying to find something in B form DNA, tension will be put on the outside to spread it apart to find active genes. It is believed that the Zform allows for easier access to active sites, and creates less tension, so proteins can access the DNA more readily. May be a cause or effect of the active genes. It is a very dynamic structure. Can change, can adapt. B form is still most physiologically important. Forms C, D, E, and PDNA have also been identified. May be important, but not sure how so at this point. o Reassociation (Reassociation Kinetics): Method of analyzing complexity of genome through separation of the double stranded DNA, and allowing it to come back together. Based on melting DNA with heat, which will break the weaker hydrogen bonds between the nucleotide bases, splitting the double helix into two single strands. The hardier, stronger phosphate bonds in the backbone will remain intact. If the strands are allowed to cool, the nitrogenous bases will snap back together, and reform the double strand. (Reassociate/renature = go back together). Reassociation can be monitored and measured with UV light – can tell how many double stranded or single stranded DNA pieces are present: The more repetitive sequences a genome has (less complexity), the more quickly the separated strands will snap back together. This is because there are more complementary strands if there is more repetition, and they do not have to match with their original partner strand. They just have to match. Can chart dissociation, and reassociation, rates: Remember, A and T nucleotides have 2 hydrogen bonds. G and C nucleotides have 3 hydrogen bonds. You have to add a little more energy to pull apart Gs and Cs due to that extra hydrogen bond. Therefore, if a genome has an A/T rich sequence, there is a lower “melting temperature” for separation of double strands. It takes less energy to denature the DNA. G/C rich strands require more energy, and a much higher temperature, to become denatured into single strands. In a full genome, dissociation and reassociation occurs in multiple steps, and therefore can be graphed in multiple curves. This is important to determine relative complexity. For instance, an organism may have a huge genome, but may simply have more repetition of genes, and fewer unique genetic sequences. In a highly repetitive genome, separated DNA strands are able to find compatible strands more quickly. Moderately repetitive genomes take longer, but still don’t have to go with their exact matches. Completely unique genomes take much longer to match up, because they have a single exact single strand to match to. Have only 1 copy of these genes sets. o Electrophoresis: DNA has a negative charge due to phosphate groups. We can utilize this negative charge to force DNA to move through a semisalad agar substance. Can put a tube of DNA into a well within an agar slab, and apply voltage. The negatively charged DNA will get pulled down to the positive end. The gel will slow the DNA down. A small piece of DNA will move very quickly and end up at the bottom. A DNA fragment that is large will not move as far. The negative charge can separate the DNA based on size. This can characterize DNA. Central Dogma: o General flow of information in a cell: DNA(replication) Transcription RNA Translation Protein. There are exceptions to this flow, for some things can reverse the process. Viral Agents – can go from RNA to DNA in a process called reverse transcription. They do this through utilization of the host cell mechanisms. Once they make DNA from the hijacked machinery, they can then use the host cell to make viral proteins. o Questions to ask about the Central Dogma: How does it work, how can it be used? Because of double stranded nature, we can see a way to replicate the DNA through template behavior. How does transcription to RNA work, and how does that allow for creation of protein? Chapter 11: DNA Organization DNA structure: o An organism has way more DNA in a cell than it has room (if the DNA is extended to its full length). So, what happens to this DNA so it can fit? For humans, if you were to stretch out human DNA in a SINGLE cell inside the SINGLE nucleus, it would measure 1.8 meters long. To cope with this space issue, the genetic material is tightly packed in different levels, making up the tertiary structure of DNA (chromatids). The primary structure is the nucleotide sequence and the phosphate sugar backbone. The secondary structure comes from the double stranded helix. In Eukaryotes, we don’t usually see DNA as a simple double helix. That is why it is called chromatin. Chromatin is a complex of DNA and proteins in eukaryotic chromosomes. o Bacterial DNA: The lowest energy state for bacterial DNA is a relaxed circular chromosome where 100 base pairs = 10 complete turns. However, bacterial cells will usually undergo supercoiling – either going to underrotate or overrotate. Adding in rotations causes positive supercoiling. Removing rotations causes negative supercoiling, the most common form of bacterial supercoiling. This is hypothesized to allow for quicker opening of the DNA for access to genetic material. Makes separation of strands easier due to expedited reduction of coil numbers. Topoisomerase is involved in this process (discussed later). Either one causes a twisting, repeating figure8 formation. Creates a much more compact chromosome. In addition, the supercoiled DNA is folded on itself with the help of proteins. o Heterochromatin: Highly condensed chromatin – Examples are inactive X chromosomes, centromeres, and telomeres – all are heterochromatic. Considered to be silent or off. o Euchromatin: This term is used to describe the DNA that we’re using, that’s active, or open, available to interact with machinery. Euchromatic DNA is found around genes that need to be used, or that we need a protein from. Chromatin o Levels of packing: Naked DNA: The simple primary structure of Nucleic Acid bases, and the secondary structure of the double helix created from the phosphate backbone. Nucleosome: First order of packing Chromatin Naked DNA will coil around nucleosomes, which are made of two tetramers of histones. There are 4 different types of histones within a nucleosome, with two copies of each (giving a total of 8 histones within a nucleosome – two tetramers (sets of 4)). This creates the “beads on a string” appearance of loose DNA. About 200 base pairs (including linker DNA) consistently wrap around nucleosomes. Without the linker DNA, it is consistently 147 base pairs. Solenoid: Second order of packing Chromatin One histone type in the nucleosomes, H1, will react with linker DNA, and is instrumental to coiling the DNA to condense it further. The solenoid shape is 30 nanometers in diameter, and usually consists of 6 nucleosomes Loop Domains: Scaffolding proteins allow for solenoids to wrap around in an even further coiled form. The scaffolding proteins lie inside the coils, keeping the solenoids folded tightly together. This structure is about 300 nm in diameter. Chromatid: The Loop domains are condensed together to form arms of a set of chromatids in Metaphase chromosomes. Metaphase Chromosome: The most condensed form of a chromosome possible, which consists of separate chromatids attached at the centromere. o Histones: Positively charged molecules that are grouped in sets of four (tetramers), which group in sets of two (to form an octamer). These form a nucleosome, around which DNA wraps. Very importantly – BACTERIA DO NOT HAVE HISTONES. They have other proteins that can interact, and condense and organize DNA. Viruses also have to compact their DNA – all their hereditary information must fit into the head alone. The virus has the advantage of not needing to use the DNA/RNA for cellular processes – it doesn’t have to be accessed due to lack of living processes. The host cell will have to use it. Chromatin remodeling: DNA must be accessible for replication, protein creation, and genetic activity. o Since it needs to be accessible, Chromatin can be remodeled. Whatever you do to structure DNA can be undone. The DNA structure must change to allow access to the genes. o Histone tails come into play here – they are excellent targets. Normally, histones bind tightly to genetic sequences, keeping them closed off. However, certain processes can be used to make them relax, making the DNA accessible. This can include acetylation, methylation and some phosphorylation. Acetylation: A process that neutralizes the positive charge of a histone to relax the histone hold on DNA. This allows genes to be access by proteins. Acetyl groups are generally considered used in active DNA, or the U chromosomes. This loosening puts genes into the euchromatin formation, looser, more accessible. Methylation/Phosphorylation: On the other hand, if you want to make DNA heterochromatic (tighter, less accessible), you might want to tighten the grip of histones with methylation and phosphorylation, forcing it to close up tightly. Google alert: Methylation – depending on what you methylate (histone, tail, body), it can have different effects depending on where methyl is at. However, in general, methylation = closing down. Chromosome banding: o We can stain chromosomes to create a banded pattern. This is a technique to quickly identify chromosomes, for the bands act as a fingerprint for the chromosome. The AT and GC rich regions of the chromosome take up stain differently. o When people were first looking at chromosomes, they were comparing their sizes, centromere locations, and other similarities. However, it can be difficult to distinguish one from the next based on location of centromere only (For example, all of the dog chromsomes are acrocentric). Banding patterns allow you to identify a chromosome, and maybe even a translocation (movement of genes of one chromosome to a separate nonhomologous chromosome). Banding can also detect an inversion or duplication within a chromosome. o There are different banding techniques – R banding, Q banding, different types of stains (UV and more). Regardless of staining or banding procedure, the banding will be the same across every individual’s chromosomes within a species, unless there is a rearrangement of some type. Specialized chromosomes: o There are a few kinds of specialized chromosomes, but they are very rare. By studying these chromosomes, we could start understanding other chromosomes. o The Polytene Chromosome: This special chromosome is visible during interphase, unlike virtually every other chromosome, so it can be monitored throughout the cell cycle. A Polytene chromosome is a replicated homologous chromosome set that divides many many MANY times, but never separates. This was discovered because the banding patterns are the same all the way across the chromosome. Function of a Polytene chromosome: The most likely goal of these large chromosomes might be that they are present during times of a dramatic change: Larval organisms about to move to the next stage Growing Plants Maybe during these stages, the organism needs a lot of a particular gene product. Polytene puff: A region of a Polytene chromosome that is relaxed and undergoing chromatin remodeling, resulting a large amount of gene activity. Can get a ton of product really quickly. Each band in a polytene region is a chromomere. o Lampbrush chromosome: Meiotic chromosome – only divides for meiosis. They get their name due to the strands of DNA pulled out from the central axis – look bristled. They exist because during division, when a chromosome is mostly compacted, we need to use some of this DNA to create gene products. This allows for Gene activity and Chromatin remodeling, which may not be possible otherwise. Centromeres: As a reminder, centromeres are the point at which sister chromatids are connected, where the spindle fibers connect during mitosis/meiosis, and do not often undergo crossing over during prophase of meiosis I. o Point centromeres – small, single point on the chromosome. o Regional centromere – This type is seen more in plants and larger eukaryotes (animals). They take up a very large part of the chromosome. For humans, one centromere might be 1000 base pairs. o Within the centromere are critical sequences called CEN regions. If you mess up these gene sequences, the centromere can no longer function, and the genome will fall apart. Chromosomes may get lost. CEN region differs from one organism to the next. For example, it is an overall region in yeast on the point centromere. It’s about 120 base pairs long. But even within this, if there is a change, it can cause a massive difference. o Centromeres are very repetitive in genetic nature. They contain many of the same nucleotides in a row. This repetition is true both within a chromosome, but also across multiple chromosomes. Within an individual, all of the chromosomes will have matching, or very similar, centromeres. o Centromeres can be millions of base pairs, and are very condensed (heterochromatic). This allows centromere staining, giving us an easy way to determine centromere location. Telomeres: o Telomeres serve as a cap to block unraveling of chromsomes. Replication of the ends of chromosomes is difficult issue, for if they replicate every time, the telomeres will constantly shorten with every division. Eventually they would be so short, they would reach a critical point causing degradation of the chromosome. We need to have a way to lengthen telomeres. There is an enzyme that replaces the ends of telomeres called Telomerase. This allows for the extension of telomeres due to the repetitive nature of telomere nucleotide bases. The telomerase contains an RNA template that will allow for replication. o Heterochromatic ends of chromosomes – transcribed regions – TERRA (Telomeric repeatcontaining RNA) – contribute to methylation, play a part in toughness for protection.) o Cancerous cells are often found to contain telomerase. Most somatic (normal body cells) do not contain telomerase, and the shortening of telomeres contributes to the aging process. Since cancer cells contain telomerase, it contributes to their immortality. However, we can use this knowledge to specifically target treat cancers. Repetitive sequences: o Most genes are unique, but some gene families can be created to make middle repetitive containing tandem repeats or interspersed retrotransposons. o Prokaryote genomes don’t have much repetition. As you go up chain of complexity, you see more and more of the repetitive information, and nongenic information. In the human genome (all 3.2 billion bases), only 2% of the genome actually makes protein. Many regions of the genome help other sections work properly, even though they don’t make proteins. We see multiple copies of genes with very important gene sequences. Also, molecular markers can be seen in minisatellites and micro satellites. They are repetitive sequences – within 1 microsatellite there are multiple copies, and the same type of microsatellite is found throughout the genome. o Highly Repetitive DNA: Includes Satellite DNA o Middle Repetitive DNA: Includes Tandem Repeats and Interspersed Retrotransposons. Tandem Repeats: Multiple Copy Genes: rRNA genes MiniSatellites: VNTRs MicroSatellites: STRs Interspersed Retrotransposons: SINES: Alu LINES: L1 o Transposons (will go more in depth in chapter 14): One of the most heavily involved factors of mutations. Transposable elements, also called “jumping” genes, are segments of DNA that can move in the genome. About 50% of our genome has arisen from these transposable elements. Retrotransposons: work through an RNA intermediate. Go through process of reverse transcription – an example of an exception to the central dogma (along with viruses). SINE = short interspersed element – Alu family, most common in humans (greater than 5% of genome). LINE – long interspersed element. Pseudogenes (look identical to other genes, but do not function properly) – adds repetitiveness to our genome. Multigene families that arise through duplication, similar idea to pseudogenes. However, while pseudogenes don’t fully function anymore. Can wipe out function, but looks pretty much identical to another gene. o Reassociation kinetics – Remember, highly repetitive regions can be indicated by using reassociation kinetics, where you apply heat to genetic material to separate double strands. Then, the single strands, as they cool, will snap back together. A modified version of this is chromosome painting. Chromosome Painting: Can utilize this to observe satellite DNA that makes up centromeres and other regions of the DNA. We can add a probe when the strands are separated – (add a piece of DNA or RNA to see how it sticks to the double strands). This probe can be labeled with different colors for different chromosomes. Literally “paints” the entire chromosome that color. Can use to see translocations, also, for instance seeing a spot of blue on a green chromosome. Can take chromosome paints from one species and paint the chromosomes of a different species, which allows us to follow evolutionary trends. GEN 3000 – Exam 2 2016 Study Guide Chapters 10 and 12 Dr. Tsai, Clemson University Exam 2: Chapters 10 and 12 Modeled around Dr. Tsai’s recommended subjects to study – color coded/organized by chapter. Chapter 10: DNA Replication Note: Unlike most everything else we study in Genetics, we can use the word ALWAYS in these chapters. Not everywhere, but in some ideas. Original Hypotheses of DNA Replication: o Conservative Replication: This hypothesis stated that the entire original double strand is conserved. After 1 replication, both strands of the original stay together, and there are two strands of new together. Then, in the second replication, have original still together, and there are 3 sets of entirely new, replicated strands. o Dispersive Replication: The idea that replication occurs in pieces, creating a patchwork of original and new replicated DNA in the new product. Semiconservative Replication: The idea that the new product conserves a single strand – Have original double strand, and those two original strands split to act as templates for new strands. The new product consists of one original strand, and one new, replicated strand. Messelson and Stahl: o These two scientists set up an experiment to see how DNA replicates in bacteria. It was based on the fact that DNA contains nitrogenous bases, so it would take up Nitrogen to build these bases. Messelson and Stahl allowed DNA to take up heavy Nitrogen ( N) or 15 14 light Nitrogen ( N), which would separate in a vial of liquid differently due to the different number of neutrons. o First, they put bacterial cells where the only nitrogen source was only heavy nitrogen. Cells incorporated heavy nitrogen into DNA. Allowed all bacteria to take up heavy Nitrogen. Then, when cells were moved to media containing light nitrogen, the bacteria then incorporated N. o When the DNA from cells were put into a tube of liquid, they found they had DNA settle in the middle, not toward the top where it would be if it was made of all light nitrogen, and not at the bottom where DNA with all heavy nitrogen would be. This suggested that the DNA strands were half old and half new. This finding allowed them to rule out conservative me15od, which they would have expected to show that DNA containing N only stayed intact after replication takes place, giving some DNA at the bottom. In addition, there would be DNA containing only light nitrogen at the top, and nothing in the middle of the tube. This DID NOT HAPPEN. o This did not rule out dispersive, so they did another round of replication in light Nitrogen media, and got a hybrid molecule, as well as DNA containing only light Nitrogen. As more rounds of replication were allowed to occur, the small amount of hybrid DNA persisted, and the DNA containing light Nitrogen built up at the top of the tube. This allowed them to rule out dispersive. Dispersive would have resulted in a gradual movement of DNA toward the light end, but always keep some of the heavy nitrogen due to chopped up pieces of original strands. They did NOT GET THIS. o These findings led them to conclude that Bacterial cells replicate in a semiconservative fashion. Old strand serves as template for new strand. Semiconservative replication: o The process of DNA replication in which a strand of original DNA is used as a template to create a strand of new DNA. The resulting double helix DNA is made of a single original strand, and a single new strand. Messelson and Stahl showed this to be true for Bacterial cells, but another experiment was required to prove it for Eukaryotic cells. o Thymidine Experiment: Eukaryotic cells were allowed to replicate in the presence of thymidine – after a round of replication, saw that both sister chromatids were labeled. Then, they followed this through anaphase without thymidine present, and saw division with chromatid separation. In the next generation, without thymidine present, they found that only 1 strand would be labelled, and the other (whole) strand would be unlabeled. This proved Semi conservative replication for eukaryotic cells. Fun Fact Experimenters were able to see was the exchange between sister chromatids in meiotic interphase due to the labelling differences. Normally, we can’t see this exchange due to identical genetic information. Replication of DNA: o All organisms use semiconservative replication, but there are differences in replication types across organisms. o Theta, rolling circle, linear: Theta: Prokaryotes have the singular circular chromosome – use theta replication. A chromosome has to have an origin of replication, there is ALWAYS an origin of replication where the chromosome will begin unwinding, where replication will begin. If a chromosome loses this origin, it cannot be replicated. When the origin opens up, there is a “bubble” formed called the replication bubble. There is bidirectional expansion – the replication bubble will expand out from between the two replicated strands. This is what gives the circular chromosome a theta appearance (Θ). There are some prokaryotes that use unidirectional replication, where the bubble opens in one direction. However, the replication is most commonly bidirectional. With bacterial replication, since there’s just a single origin, the entire organism is called a single replicon. A replicon is the newly replicated fragment from one origin, and the number of bases replicated in one set. For E. coli, have a single origin, so a single replicon of 4,600,000 base pairs. Rolling Circle: Another mechanism that viruses and the F factor replicate by is the rollingcircle replication. Instead of a bubble forming at the replication origin, the strand will actual cut and peel away as it is replicated. The cycle may be repeated. Do NOT see a replication bubble because this process is unidirectional. Linear: In eukaryotes, there are multiple replication bubbles that open up, and are all bidirectional, moving away from the origin. There are multiple origin points due to the extensive amount of DNA to replicate. Therefore, there are multiple replicons, or segments of DNA replicated in one go. o Origin: The place at which replication begins. There ALWAYS must be an origin of replication in order for genetic material to be replicated. If the origin somehow malfunctions, or is lost, replication will not take place at all. Typically, an origin (regardless of the type of replication taking place) is a distinctive sequence of base pairs. DNA is antiparallel, we can only make DNA in a 5’ to 3’ direction. Bases will be added in the opposite direction of the strand. In a bidirectionally replicating DNA strand, one strand will synthesize away from fork, and one towards the fork, where the origin of replication is located. Lagging Strand: As the fork is moving, the strand moving away from it has to wait until the fork moves to add more nitrogenous bases, forcing it to replicate in fragments (called Okazaki fragments). Leading Strand: The other strand, moving towards the fork will continuously synthesize in one single piece. No fragments are created. o Steps: Opening and Stabilizing the DNA Strands: In order for replication to occur in semiconservative fashion, the double helix of the original DNA strand must be “unzipped” or opened for replication. Helicase comes in and unzips the DNA, breaking the hydrogen bonds. Once you start separating the DNA, it wants to snap back together, so you have single stranded DNA binding proteins that keep the single strands from coming back together. Helicase always located at the fork of the replication bubble. DNA gyrase (prokaryotes) is always present ahead of the fork to relieve the tension formed in the rest of the DNA strand. o Topoisomerase: Gyrase is a type of topoisomerase in prokaryotes. Eukaryotes do not have gyrase, just have topoisomerase. Priming the Strands: Priming is required for DNA synthesis. DNA polymerases cannot initiate DNA synthesis without a 3’ –OH group to extend. The Enzyme Primase will provide 10 – 15 nucleotides as a primer for polymerase activity. This primase is going to put down RNA instead of DNA. Anytime the polymerase is going to synthesize DNA it has to have that primer first. On the leading strand, you need a single primer. Once polymerase says it has the –OH group, it continues on with the replication fork. It doesn’t need another primer. On the lagging strand, every time a new fragment needs to be made, a new primer is also needed. Elongation of RNA Primers (laying down complementary nitrogenous bases): Now that primer is present, the DNA can be made. E. coli has 5 DNA polymerases: o DNA polymerase I was the the first discovered – it has 5’ to 3’ polymerase activity. It can also do 3’ to 5’ exonuclease activity – allows correction of errors (exonuclease = getting rid of DNA). Also has 5’ to 3’ exonuclease activity that is used to remove primers. The main function of this polymerase I might be to remove the primers. Also called the Kornberg polymerase. o Polymerase III is a large complex, has 5’ to 3’ normal polymerase activity as well as corrective 3’ to 5’ exonuclease activity. This is the main one that lays down the nitrogenous bases (DNA) complementary to the parent strand. o Polymerase II – can do 5’ to 3’ and 3’ to 5’ and is involved in DNA repeair, and restarts replication after damaged DNA halts synthesis. o Polymerase IV: Involved in DNA repair. o Polymerase V: DNA repair; translocation – DNA synthesis Removal of Primers: Once replication is complete, the RNA primer gets replaced by DNA bases through the action of polymerase I. Joining of Okazaki fragments: DNA ligase comes in and seals the fragments. The Okizaki fragments also undergo this. Termination: In some organisms, there isn’t a specific thing that causes termination. Once the forks meet, the process is stopped. Other organisms have termination proteins that stop replication. Has one way directionality. Tus/ter It can only go through a replisome (replication machinery) in one direction. Want to make sure it starts at the origin, circle all the way around, and stop. The replisome is all of the replication machinery. Replisome – has the Pol 3 primer, the helicase, etc. It is incredibly fast, almost replicating at 1000 bp/second, and is doing so incredibly accurately. It is a very fast, accurate process. o Required proteins/enzymes: Helicase: Unzips DNA Single Stranded DNA Binding Proteins (ssDNA BP): Proteins that keep the protein from snapping back together after the bubble is opened. Pimase: Puts down an RNA primer Gyrase: Relieves tension ahead of the fork in prokaryotes Topoisomerase: Relives tension ahead of the fork in eukaryotes Polymerase I: 5’ to 3’ AND 3’ to 5’ activity to remove primers and lay down DNA. Polymerase II: 5’ to 3’ and 3’ to 5’ activity for proofreading. DNA repair. Restart replication. Polymerase III: 5’ to 3’ normal activity and corrective 3’ to 5’ exonuclease activity. Main DNA synthesis compound. Polymerase IV: DNA Repair Polymerase V: DNA repair, translocation. o Directionality: Replication, Transcription, and Translation ALWAYS proceed in the 5’ to 3’ direction, which means that they are reading the parent strands in the 3’ to 5’ direction. MUST BE CAREFUL OF THIS ON THE EXAM. There are a few, rare exceptions to this directionality, such as viral replication, or proofreading mechanisms. o Proofreading: Various polymerases have proofreading capability (I, II, and III) – This is the exonuclease activity, 3’ to 5’ activity, that will remove, and replace, incorrect bases that may be laid down. THIS IS ESSENTIAL TO CORRECT DNA REPLICATION ERRORS. o Eukaryotic differences: The eukaryotic system is very similar to prokaryotes. Overall, the system works the same. There is a single stranded template, primer, polymerase, needs 3’ OH, etc. Everything is a little more complicated with Eukaryotes. One, we have more than one origin. Two, we have more polymerases. Three, nucleosome assembly makes things more difficult. Four, they are linear chromosomes. Origins: There are many different sequences along the chromosome that signify that it is an origin of replication. In yeast, these are called ARS, autonomously replicating sequences. Everywhere you see the sequence, regardless of its location on the chromosome, or which chromosome they’re on, they look essentially, or exactly, the same (consensus sequence). This helps to identify all origins. In mammals, it appears we don’t need a consensus sequence, but there is still a similar chromosome structure (focuses more on tertiary structure rather than nucleotide bases). Licensing factors: With all these different origins, how do we make sure we don’t overuse a particular origin? Licensing factors – essentially give a license to an origin that allows it to start replication. It gives the origin the necessary items to start replication. These factors recognize origins and bind during the G1 phase, prior to DNA replication. These proteins will find and bind to all origins, essentially flagging them so that, in S phase, the replication machinery can find them. o Once they’re gone, we can no longer recognize that origin, and we cannot start replicating there again. One time use. Unwinding: Once you get initiation going, eukaryotic replication looks virtually the same as prokaryotic. Helicase break hydrogen bonds, which are stabilized by single strand binding proteins. Have topoisomerases outside of the replication fork to relieve tension, instead of gyrase. Primase then comes in, lays down the RNA primers. Specific polymerases: In eukaryotes, polymerases are more specified in their functions. DNA polymerase III handles lagging and leading strands in prokaryotes, but there are special ones for each specific type of DNA, and the leading and lagging strand, in eukaryotes. In addition, there are many more polymerases involved in repair to DNA. There is a lot of machinery dedicated to fixing any issue that comes up. Eukaryotic has proofreading ability to backup and fix mistakes, even more so than prokaryotes. Nucleosome Assembly: Disassembly and Reassembly of nucleosomes takes place right before and after the replication fork. Chromatin remodeling is so important. Nucleosomes are present right up until the old DNA is being replicated, and then you see them immediately form after replication. Only unwinds right around the fork. It is so important to open up chromatin and to reclose it when needed. Essential. o Linear problem: Unlike prokaryotes, which have a circular chromosome that meets at each end after replication, a linear chromosome must simply stop at each telomere. How does this work? We have to put the RNA primer at the end to make DNA, so we have to be able to remove it. When we remove the RNA primer, it leaves a gap at each end of the chromosome since there is no 5’ end to attach to, unlike prokaryotes. All of the primers in the middle are not a problem due to the meeting of 5’ and 3’ OH groups. If the gap does not get filled, we have constant shortening of the telomeres. If they get shorter, we see degradation of the chromosomes. So, we have to have some way to finish the replication strand. Telomerase comes into play. Telomerase is a ribonucleoprotein, and is complementary to the DNA strand. The RNA template will bind to overhang of the longer, parent strand. This provides a template for new bases, which allows for the availability of a 3’ OH group. We can add on bases that can match up or are complementary to the new DNA. It can do this repeatedly as needed to fill in the gap of the telomere. We then have extra DNA on the end of the telomere, extending it. NOTE! We can only do this because the telomere is repetitive, and the repeats get added again and again. There will still be a gap, but even though the gap is there, we have extended the DNA beyond the original end, and it will be unaffected by the gap. The overhang of extra DNA can then fold over on itself to see nonconventional base pairing (such as a TT or GG). It can now provide a 3’ OH group that lets it be filled in during replication. Regardless, the gap gets filled, and the telomere may be extended. Telomerase is active in our germ line. We do not want to pass on preshortened telomeres. Most somatic cells DO NOT have telomerase. So, in skin cells, or other somatic cells, the telomeres shorten continuously. This is involved in the aging process. Chapter 12: Transcription and Translation RNA vs DNA: o RNA is typically single stranded, while DNA is typically double stranded. The single stranded nature of RNA allows it to acquire more unusual shapes, while DNA is pretty much locked in its position. o RNA contains a ribose sugar instead of a deoxyribose sugar. o RNA contains Uracil instead of Thymine as a Pyrimidine Base. This pairs U and A together. o We have decided that RNA that probably came first in the evolutionary line because RNA can more commonly be catalytic. Thomas Czech in 1981 discovered the Hammerhead ribozyme – it is catalytic RNA (carries genetic information), but it can act like a protein without having any protein elements. o Since this discovery, we have found that there are other ribozymes (genetic material that can act as enzymes/proteins). mtDNA primase is one (ribonucleoprotein complex). RNA was likely the original genetic material and could carry out primitive catalysis. It probably switched to DNA because it is much more stable than RNA. o Differences between Eukaryotes and Prokaryotes: Prokaryotes have somewhat “exposed” DNA due to lack of nucleus. Translation/transcription can all happen in the single location. In eukaryotes, on the other hand, transcription and translation require proteins to be made in the cytoplasm. Transcription takes place in the nucleus, then it undergoes processing of RNA, which prepares RNA to travel to the cytoplasm for protein creation. Types of RNA: o Prokaryotes: Messenger RNA (mRNA) that carries the code for proteins. Ribosomal RNA (rRNA) Transfer RNA (tRNA) These are all three found in eukaryotic and prokaryotic organisms. o Eukaryotes also have many other RNA types: Premessenger Small nuclear Small nucleolar Small cytoplasmic MicroRNA Small interfering RNA Piwiinteracting RNA. Transcription o Requirements: ssDNA template: Need to unwind DNA so it can be red Substrates to make RNA (ribonucleoside triphosphates!): Two triphosphates get cut off to provide energy for the sugarphosphate backbone. With the triphosphates, it will still dictate direction. We make RNA from 5’ – 3’. This means you are reading DNA 3’ – 5’ because it has to be made antiparallel to DNA. Transcription machinery: RNA polymerase is a huge player in transcription. Handles the majority of the work. Unwinds the DNA as it moves through. Rewinds it as it passes by. Transcription bubbles open and allow us to have the ssDNA template. o NOTE: Transcribing only occurs on one strand – important difference from replication. It is selective in the fact that only one single template strand from a double strand of DNA Is needed. You can see a chromosome being replicated on each strand individually in different directions. Transcription often occurs more than one time for a single gene. As polymerase moves through on one gene, another polymerase can follow and start transcribing again immediately after the first. This gives a “Christmas tree” effect – this allows more than one protein product for each gene to be produced at a time instead of producing proteins one at a time. o Directionality: Transcription still must occur in the 5’ to 3’ direction, meaning it is reading the original strand 3’ to 5’. o Template vs nontemplate: Template Strand: There is one strand being actively read and transcribed – the template strand. The template strand is complementary and antiparallel to the RNA being produced. It is also complementary to the nontemplate strand. NonTemplate Strand: The other is a nontemplate strand, also called the coding strand. The RNA looks very similar to it. This allows us to quickly determine what the mRNA will l
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