BIO Study Guide Notes for Exam 2 PDF

CHAPTER 20: Applications of DNA Replication- PCR and DNA Sequencing The Polymerase Chain Reaction (PCR) PCR is the amplification of a specific DNA region that does not require a cell....

CHAPTER 20: Applications of DNA Replication- PCR and DNA Sequencing The Polymerase Chain Reaction (PCR) PCR is the amplification of a specific DNA region that does not require a cell. How does this work? We need… -​ A wanted DNA portion that we want to replicate. -​ Buffer -​ Things that are necessary to make more of that DNA -​ Primers, DNA Polymerase (Heat resistant ex. TAQ Polymerase), DNA Nucleotides to build with. PCR Sequence 1.​ Denaturation -Heat is added to separate the DNA strands from one another. 2.​ Annealing -When the two DNA strands that have just been separated are cooled down. The temperature allows the primers to attach to whichever sequence of the DNA you want to replicate. 3.​ DNA Synthesis -DNA Polymerase starts at the primers and uses the DNA nucleotides to create the complementary strands of the original strands. -This results in two double stranded DNA molecules. This can be done over and over again to make thousands of copies of the same DNA sequence. How to design the primer needed for a PCR reaction? Some important factors must be considered when designing a primer for PCR… Primer Length -Between 18-24 Base Pairs Annealing and Melting Temperature -Annealing temp should be 5 degrees below the melting temperature. Secondary Structure -Be sure that the secondary structure does not cause the primer to fold in on itself instead of attaching to the DNA sequence you want to amplify. Exponential Amplification For each round of PCR there are two double stranded DNA molecules created. Therefore for any n amount of cycles there are 2n DNA Molecules. Gel Electrophoresis of DNA This process is used to separate charged macromolecules based on their size. An electrical current is run through a type of gel. This gel is either made of agarose or polyacrylamide gel. -Agarose is a polysaccharide polymer (a carbohydrate from seaweed) which lets DNA molecules travel in it. It has large pores which helps for separating larger molecules like DNA. -Polyacrylamide Gel is another gel that has smaller pores which makes it more useful for other molecules like proteins. Process of Gel Electrophoresis 1.​ DNA is cut by restriction enzymes. 2.​ DNA is placed into a well that is negatively charged. The opposite end of the gel is positive. Since the DNA is negative it will travel towards the positive end. 3.​ The longest fragments of DNA are heavier and take longer to travel so they are closer to the top of the gel. The shorter fragments are lighter and therefore will travel further down the gel. 4.​ The only good way to see the DNA bands is to stain the gel and view it under a UV light. Sanger Sequencing This technique identifies the order and type of nucleotides in a DNA segment. DiDeoxy Nucleotides (dNTP) -This if the hydroxyl group is removed from the 2n and 3n side of the sugar then it is a dideoxynucleotide. -A deoxynucleotide is when only one of the hydroxyl groups is missing from the central sugar. -DNA polymerase uses these as a substrate. The way that DNA is connected is through a reaction between this hydroxyl group (3n) and the phosphate of another nucleotide. So without this group present the nucleotides cannot connect to one another during sequencing. Procedure 1.​ The template strand of DNA that needs to be sequenced is divided into four tubes. 2.​ Each tube contains a primer that will bond with the template strand of DNA. These four primers are…  -dATP, dTTP, dGTP and dCTP 3.​ DNA polymerase adds the dNTPS onto the template DNA and carries out regular polymerization. 4.​ Each tube will also have a radiolabeled dideoxynucleotide at very low concentration such as… -ddATP (Tube1), ddTTP(Tube2), ddGTP(Tube3), and ddCTP(Tube4). 5.​ This causes each of the fragments to show their length wherever they terminate and locate that specific nucleotide in the sequence. 6.​ Then in combination with gel electrophoresis and an X-Ray, the sequence of the DNA starts from the ‘bottom’ of the film and can be read upwards. Modern Sanger Sequencing 1.​ Each ddNTP is marked with a florescent dye so each tube has a different color when exposed to UV Light. The dyes include… -​ Red, Blue, Yellow and Green 2.​ This allows for the reaction to be held in just one tube instead of four since all the ddNTPs appear as different colors. 3.​ This also means that when a polyacrylamide gel is used with the gel electrophoresis then it will only show up in one out of the four columns. Therefore the one on the right would be the old method of sanger sequencing with the different nucleotides separated into different columns. The one on the left would be modern sanger sequencing where they are all included in one of the wells using gel electrophoresis and fluorescent dyes. 4.​ Modern Sanger Sequencing uses what is known as capillary electrophoresis which includes CCD (charged coupled device) for the detection of fluorescent signals. CHAPTER 17 AND 18.1 Prokaryotic Transcription/Translation and Gene Regulation:Operons The Central Dogma The information within DNA is transferred to mRNA (messenger RNA) which then directs the synthesis of proteins. This set of instructions encoded within the DNA is called the genetic code. Properties of the Genetic Code ​ Unambiguous: It directly codes for amino acids. ​ Triplet-based code: DNA is organized into codons which are triplets of nucleotides that code for a specific amino acid. ○​ If there is a shift in the frame (an addition or subtraction of a nucleotide or codon) then the entire frame is shifted since each codon cannot code for the right amino acid. ​ Redundant Code: There are only four nucleotides that code for all amino acids therefore the code is redundant. ​ Universal Code: It is almost universal meaning that most organisms use the same codon to code for the same proteins but there are variations in things like mitochondrial RNA that make it not universal. ​ Non-overlapping: There is a reading frame for DNA where each triplet of nucleotides is read as a codon. If there is a mutation that causes the addition or subtraction of a nucleotide that will cause a frameshift mutation and a non usable protein will be translated. Transcription 1.​ DNA is used as a template to synthesize mRNA. 2.​ So the RNA polymerase will create a complementary strand to the DNA sequence that is needed to create whatever protein is needed. Remember this strand will not include T’s only U’s since it is still an RNA strand. 3.​ This mRNA has to undergo splicing (more of this within the next chapters) to make sure that all the introns are taken out and ligase connects the exons so that the protein is directly coded from the mRNA. 4.​ The mRNA then leaves the nucleus. Requirements for Translation ​ mRNA: Contains the message ​ Ribosomes: Responsible for making proteins ​ tRNA’s, Amino acids and tRNA aminoacyl synthetases ○​ tRNA aminoacyl synthetases are enzymes that attach amino acids to tRNA in the first step of protein translation. ​ Elongation factors: Aid in translation ○​ They carry tRNA’s to the ribosome’s A site. ​ Energy in the form of GTP ○​ This is to move the ribosome along the mRNA strand as it codes. Translation 1.​ The mRNA travels to a ribosome (rRNA). 2.​ The small subunit of the ribosome with a tRNA and initiation factors binds to the 5’ end of the mRNA. 3.​ The complex scans the mRNA from the 5’ to 3’ end until it reaches the start codon which usually is AUG. -mRNA binds noncovalently to ribosomes. 4.​ tRNA binds (noncovalently) to the AUG (start codon) at the initiation site and recruits the large subunit of the ribosome to start the process of translation. 5.​ Codons on the mRNA bind to complementary sequences on the tRNA (transfer RNA) which has an anticodon and contains the amino acid that the codon codes for. -EXAMPLE: If the Codon on the mRNA is AUG then the anticodon on the tRNA is UAC. 6.​ The amino acid clips off when the tRNA and mRNA bind to each other. The amino acids are then bonded together with a peptide bond. 7.​ When the stop codon is reached by the ribosome, release factors bind to the stop codon which dissociates the mRNA from the ribosome. -REMEMBER: The stop codon DOES NOT code for an amino acid. Therefore if there are 3000000000000 codons for a protein there will only be 299999999999 amino acids. 8.​ Protein folding and modifications occur after the new amino acid sequence is released from the ribosome. \ Regulation of Gene Expression -RNA based processes that alter the structure of the DAN molecules without changing the sequence can inhibit or promote the transcription of certain genes. (More of this to come in later chapters when discussing operons) -Once a gene is transcribed, translation can be inhibited by small regulatory RNAs stopping the gene from being translated into a protein. Eukaryotes As always eukaryotes are different from prokaryotes when it comes to different processes. Here are some of those differences. -​ Before an mRNA can be translated by the rRNA there needs to be changes to the sequence that occur within the nucleus. These changes include… a.​ Splicing-Introns (noncoding regions) are removed from the mRNA and exons are stuck together by a spliceosome. i.​ Spliceosomes detect sequences at the ends of introns, cut them out and then connect the remaining exons. b.​ At the 5’ cap there is a 7-methylguanosine (a modified guanine nucleotide) attached to the first nucleotide by a triphosphate linkage. i.​ This cap is what is recognized by the ribosome and where it binds so that translation can start. c.​ At the 3’ cap there is a polyadenylation signal that recruits a RNA bind protein (CPSF) that directs an endonuclease to cut the transcript at the 3’ end. Polyadenylate Polymerase then added a long string of adenine nucleotides to the 3’ end which is then called a Poly-A Tail. -​ These modifications are needed for proper coding, avoid degradation of the mRNA by enzymes and signal that the mRNA is ready to leave the nucleus. -​ Transcription occurs in the nucleus and translation occurs in the cytoplasm. Prokaryotes As always prokaryotes are a bit different because they have a different organization for DNA than eukaryotes. Regulation of Transcription in Prokaryotes When a gene in a prokaryote is to be transcribed then the RNA Polymerase will bind to what is called a Regulatory DNA Sequence. -Unlike eukaryotes which have one promoter per each gene, prokaryotes can have one promoter for multiple genes. Promoter:The regulatory DNA sequence where the RNA polymerase binds to start transcription. Represser: Attaches to the DNA after a promoter to block transcription. -There are some situations where the repressor can only repress when another molecule is attached to it. This other molecule is called a corepressor. Operator: Regulatory DNA sequence where the repressor attaches to block transcription. Activator: Binds to a regulatory sequence so that there is even more transcription than a normal trickle level. -There are also some small molecules that turn the activator on that are called inducers. These act in the same way as corepressors. Tryptophan Operon This operon is present in E.Coli. The enzymes that are made through this operon are used to make tryptophan. Generally, this is on by default but can be turned off with the use of a repressor. This operator contains a… -​ A promoter -​ A repressor -​ A operator -​ A corepressor In a low tryptophan environment that means WE NEED MORE TRP! The only way to make more trp is to transcribe the genes and make the enzymes that make trp. Therefore the rna polymerase will clip onto the promoter so that transcription occurs. In a high tryptophan environment then that means WE DON'T NEED MORE TRP! Therefore trp acts as a corepressor for its own operon to prevent transcription. -​ There is another way to stop the production of trp, which is called Feedback Inhibition. This is when trp acts as a competitive inhibitor which disables the very proteins that produce it. Lac Operon This is an operon that is present in E.Coli. The enzymes that are made help with the breakdown of lactose into glucose and galactose. LacZ -​ Codes for an enzyme that turns lactose into simple sugars LacY -​ Codes for an enzyme that allows for the absorption of lactose through cell membranes. LacA -​ Basically is a helper to the other genes. LacI -​ The repressor for this operon. CAP Site: This is where a Catabolite Activator Protein will bind and activate to increase transcription. caMP is suppressed when glucose is around but when there is no glucose then there is nothing suppressing it allows for activation to occur. No lactose- The lac repressor is bound to the operator to avoid any enzymes being transcribed since there is nothing to break down. High Lactose- The lac repressor is inactivated by allolactose which is an isomer of lactose and no longer blocks transcription so that the gene can be transcribed. Lactose can be metabolized. But… glucose is preferred over lactose. No Glucose + High Lactose= Transcription occurs High glucose No Lactose= Transcription does not occur No glucose and no lactose= Transcription does not occur. High glucose + High Lactose=Low caMP won't be able to bind to the CAP site and there will be less transcription. Translation: 1.​ Before translation can start the ribosome must bind to the mRNA. The rRNA binds to something called the shine-dalgarno sequence which is the binding site that helps initiate protein synthesis in prokaryotes. 2.​ Then after another noncoding sequence we reach the start codon which in prokaryotes is formal methionine (so a methionine with a formal group attached). a.​ This acts as an alarm system in the human body so that the immune system can be activated to kill the bacteria that is present inside of our bodies that may cause infection. 3.​ Translation occurs as normal until it reaches the stop codon. 4.​ Then a noncoding region is hit again. Prokaryotes do not have a Poly-A Tail or 5’Cap because transcription and translation occur in the same place and can happen at the same time so there isn't a chance for degradation. BOTH EUKARYOTES AND PROKARYOTES HAVE POLYRIBOSOMES -​ Polyribosomes are groups of ribosomes that work together to translate mRNA into polypeptides. This happens even before the actual mRNA is finished being transcribed. Coupling of Transcription and Translation Basically the idea that transcription and translation are both separated in eukaryotes by the nuclear membrane and one cannot occur without the other being completed first. However, in prokaryotes since there is nothing to separate the two actions then they can be coupled and occur together in the same place and also at the same time. Effects of Mutations on Translation -​ Substitution -​ Silent Mutation: A nucleotide is swapped for another nucleotide but that same codon still codes for the same amino acid. Therefore the protein that is made is still fully functional. -​ Mis-sense Mutation: When there is a DNA change that replaces an amino acid sequence with a different amino acid sequence therefore the wrong amino acid is coded for. Most likely as long as a stop codon is coded for this doesn't cause that many issues. -​ Nonsense Mutation: When a change in a nucleotide or codon produces a premature stop codon leading to a shortened version of a protein's amino acid sequence. Most likely this causes the protein to not work or be defective. -​ Insertion or Deletion of a Nucleotide or Codon produces a frameshift mutation and is more likely to produce a defective protein. CHAPTERS 17.3 AND 18.2 Eukaryotic Genome Structure/Regulation 1.​ Eukaryotic Gene a.​ Distal Control Elements: DNA sequences that are far away from the genes that they regulate. b.​ Proximal Control Elements: Regulatory DNA sequences that are located near and promoter and control gene expression. c.​ Promotor: The point at which RNA polymerase binds so that the transcription of the gene that the promoter controls can take place. d.​ Gene: A sequence of DNA that codes for a particular protein. Now DNA has both coding and noncoding regions therefore before translation can occur RNA splicing must occur in the nucleus. This is so that the mRNA directly codes for the protein that is needed. e.​ Poly A Sequences: A long chain of adenine nucleotides. f.​ Terminator: A section of nucleic acid sequence that marks the end of a gene during transcription so that the RNA polymerase knows when to stop transcribing. 2.​ Eukaryotic mRNA a.​ 5’ Cap- the 7-methylguanosine cap b.​ 5’ UTR- This is an untranslated region in the beginning of the mRNA coding sequence before the stop codon. i.​ Serves as a recognition site for the rRNA so that it can find the start codon and start translation. ii.​ Can influence the rate of translation initiation by interacting with regulatory proteins allowing for protein expression based on cellular conditions. iii.​ Complex RNA secondary structures within this region can regulate translation through ribosome scanning and access to the start codon. iv.​ Allow for control of translation in response to environmental cues. Regulation of TRanscription in Eukaryotes -RNA Polymerase 1 -Makes rRNA and is present in the nucleolus. -RNA Polymerase 2 -Makes mRNA, miRNA (micro RNA) and snRNA (small nuclear RNA). -RNA Polymerase 3 -Makes tRNA and 5S rRNA. IN PROKARYOTES THERE IS ONLY ONE RNA POLYMERASE Chromatin Structure -​ Histone Modification -​ Acetylations: Chromatin is made up of histones which have DNA wrapped around it. Depending on how tight the wrapping is the access to genes may be limited. For example, if the wrapping is too tight then the access to the genes will be low.  If it is loose then more proteins will be able to interact with the DNA allowing for interactions. -​ This takes place in the lysine residues. The phosphate group of the DNA and the NH3 group in the lysine and have electrostatic attraction between them. The additions of the acetyls weaken the histones which weakens the charge and weakens the bond making the DNA more loosely wrapped around the nucleosome. -​ Methylation -​ This also takes place in the lysine residues but instead it winds the DNA closer to the histone which makes it harder for the DNA to be transcribed. -​ DNA Methylation -​ This is important for embryonic development, genomic imprinting, x-chromosome inactivation and preservation of chromosome stability. -​ Cancer uses this. -​ This can occur either in the promoter of the gene body. -​ This can open up the gene for expression or close the gene to silence it. Works really similar to histone methylation. Transcription Initiation -​ RNAP2: Transcribes all the mRNA needed for translation. -​ General Transcription Factors and Mediators -​ GTF’s initiate transcription by binding to the promoter region of that gene which then allows RNA polymerase to bind and transcribe the gene. -​ Allows GTF to interact with RNAP2. CHAPTER 20.3 How Do We Assay Gene Expression Detecting RNA Expression -​ Fluorescent In Situ Hybridization (FISH) using RNA probe -​ Uses RNA fluorescent probes which detect mRNA transcripts within the cell allowing for us to see if, when and where a gene is being expressed within a cell. -​ RT-PCR: The making of cDNA -​ An RT enzyme and an RNA template create a single stranded copy of cDNA. -​ A DNA polymerase amplifies the cDNA into a double stranded cDNA molecule. -​ Standard PCR can then occur which will produce multiple copies of the cDNA that can be used. -​ Northern Blotting -​ Purify the RNA within a sample -​ Use an electrical current to separate the different fragments using agarose (gel electrophoresis). -​ Move the RNA onto a solid membrane and expose it to a DNA probe. This probe must be fluorescently labeled so that when the DNA and RNA interact then it can glow and we can detect if the RNA is there or not. Detecting Protein Expression -​ Use GFP tags -​ To clone/ express GFP -​ 1. Amplify using PCR. -​ 2. Use cohesive end cutters to digest the GFP product and host vector. -​ 3. Ligate the cohesive end with T4 ligase -​ 4. Transform the plasmids into competent cells -​ 5. Measure fluorescence to test expression of GFP. -​ This is for use within a plasmid to make recombinant DNA -​ To express eukaryotic genes in prokaryotic cells you must use cDNA you cannot use genomic DNA because it will not directly code for the proteins needed. -​ Use antibody -​ Mainly to detect the location of target proteins in cells and tissues -​ To determine the amount of target protein -​ Can help determine protein structure. -​ Western Blotting -​ Use a polyacrylamide gel for SDS Page gel electrophoresis. -​ This GE needs charged proteins so use SDS and beta-mercaptoethanol. For BOTH WESTERN AND NORTHERN BLOTTING YOU NEED TWO CONTROLS -POSITIVE CONTROL/ NEGATIVE CONTROL -Meant to show what a positive result looks like. For example if you are using northern blotting to detect the RNA of a bacteria in a sample then you would have a colony of that specific bacteria loaded into the blot as a positive control. This is meant to be a reference point for what your results should look like.  -A negative control is used normally as a sample but is not expected to change. This is to make sure that if it does change we aren't actually doing the procedure correctly or something else has gone wrong. It does not contain the target protein! For example if you are testing for a protein in a cell you would put the same cell of say a bacteria without that protein expressed to ensure that nothing else is changing in the experiment other than the amount of the protein produced. You also wanna make sure that the protein production is because of something else. -LOADING CONTROL -GAPDH -Tubulin -Actin -The loading control is to make sure that an equal amount of protein has been loaded into each well. CHAPTER 11- Signal Transduction Fundamentals of Intercellular Communication (Between cells) 1.​ Unicellular Organisms a.​ Bacteria use quorum sensing which is when they send signals between each other about external conditions. b.​ Yeast Saccharomyces Cerevisiae only have two mating types which are called a n alpha. They use mating pheromones to mate with the opposite type. Some yeast can even switch which mating type they are. 2.​ Multicellular Organisms a.​ A signal is sent out usually as a ligand and binds to a receptor of that logan on a target cell. That receptor detects the signal, intermediate steps occur in the cell so that a cellular response can be performed. b.​ There are ways for direct contact such as gap junctions between animal cells and plasmodesmata in plant cells. c.​ For long distance communication you can use endocrine signaling which releases ligands into the bloodstream to reach cells that are further away. d.​ Autocrine signaling is when a cell sends out a ligand that then activates on its own cell membrane to produce a cellular response. Signal Transduction -​ Steroids are hydrophobic which allows them to pass through the cell membrane and reach their very own type of receptors that are inside the cell called intracellular receptors. -​ Once they bind they do not activate transcription in the cytoplasm instead the now activated receptor is told to move into the nucleus for the transcription of whatever genes it is being told need to be transcribed. -​ Membrane Receptors -​ This is just the idea that since most signaling molecules are unable to pass through the cell membrane they must use a transmembrane receptor so that the signal can be transduced. -​ There are three types of membrane receptors -​ Receptor tyrosine kinases (RTK) -​ Only activates if two ligands bind (one per tyrosine) and then they join together to form a dimer. Then ATP is needed so that they can activate along with activated relay protein to translate the signals needed. -​ G Protein Coupled Receptors -​ Ligand binds to the GPCR -​ G Protein associates with the now activated receptor -​ Then the G protein releases GDP to bind to CTP and activate. This sends the signal out. -​ TO DEACTIVATE  -​ The GTP attached to the G protein turns into GDP and the G protein is released from the activated enzyme. -​ Ligand Gated Ion Channels -​ When activated by a ligand they open and allow things in. This changes the cell's electrical properties and that is what triggers the cell response. -​ Second Messengers -​ SM’s like cAMP and calcium amplifies cell signals. -​ This is possible because one initial signal molecule can activate the SM’s which activates multiple downstream signals overall leading to more stimulation than would have been possible with just one signal. Cell Signaling in Human Disease So basically cancer uses the Ras-RAF-MEK-ERK pathway which is where Ras (a g protein) is constantly locked on. However, cells can bypass that by inactivating the kinases that are meant to be activated by the RAS after. CHAPTER 12 Mitosis and The Cell Cycle Homologous Chromosomes look like what a stereotypical chromosome looks like. A sister chromatid is half of the homologous chromosome. The sister chromatids are held together by a centromere. When trying to count the amount of chromosomes make sure you count centromeres not the number of chromosomes themselves to make sure you get the correct number. Diploid (2n): This is the whole amount of chromosomes that each somatic cell would have unless there was something wrong. Haploid (n): This is half of the diploid number of chromosomes. This only happens in gametes. Karyotype Basically a layout of all the chromosomes an organism has to see mutations or lack of chromosomes. Euploid:Normal amount of chromosomes Aneuploid: An abnormal amount of chromosomes whether that be too many or too little Stages of the Cell Cycle Interphase: Takes up most of the cells life especially if they never have to perform mitosis after they enter the G0 phase. This is where normal cellular functions take place. G1 Phase: Growth occurs here. This is like the commitment state because after this stage if the cell is not meant to continue dividing or dividing then it will enter the G0 phase. S Phase: DNA is replicated here G2: Further preparations for cell division occur. This may technically be before the m phase but since the cross over is once anaphase occurs that means prophase and metaphase are included in the G2 part of the cell cycle. M Phase: Where mitosis occurs. The start of the M checkpoint is when anaphase occurs. If nondisjunction occurs here then that will not produce two proper daughter cells. In fact since mitosis only has one division then both of the daughter cells are messed up. Mitosis Steps 1.​ Prophase This is where the nuclear envelope and nucleolus dissipates and the chromatin condenses into chromosomes. Spindles are created within the centrioles and then attach to centromeres. 2.​ Metaphase  Chromosomes align along the cell's equator and the spindle fibers stay attached to the centromeres. 3.​ Anaphase Sister chromatids are pulled apart by the spindle fibers at the centromere. 4.​ Telophase Nuclear membrane and nucleolus reappear and the chromosomes become chromatin again. 5.​ Cytokinesis The division of cytoplasm between the two daughter cells by pinching off (cleavage furrow) in the middle. Keep in mind that plant cells use a cell plate which develops over time to then become a cell wall to divide its daughter cells. The Cell Cycle and Cancer 1.​ Chemical Factors influencing cell division a.​ Growth Factors b.​ cDK’s are activated by cyclins to move the cell onto another phase. 2.​ Physical Factors influencing Cell division a.​ Density Dependant Inhibition i.​ Basically is a cellular process where normal cells stop divindg and gorwing once they reach a particular density. This mainly happens when they come into close contact with other cells. b.​ Anchorage Dependance i.​ Normal cells can only really grow and survive when they are attached to a surface or a matrix. Cancer cells on the other hand dont need to be attached to anthying to grow and survive so thats why they tend to move around. So a cancer cell from your lung could move to your left toe only becuase cancer cells dont need to hold onto anything to survive. 3.​ Cancer cells continue to divide because they add telomeric sections to the end of the DNA during replication, by pass the signals that are needed for the cell cycle and are anchorage independent.