Microbiology Study Guide Lecture Test 3 re Z Chapter 8 | Segments of DNA that code for functional products. | Function Enzymes An organism's genetic makeup. Information that codes for all the particular characteristics of the organism. The genotype represents potential properties, but not the properties themselves. | Actual expressed properties of the organism, such as the | organism's ability to perform a particular chemical reaction.In a | sense the phenotype is a collection of proteins. | Ribozyme- RNA enzyme that removes introns and splices | exons together | Photolyse- uses visible light energy to separate UV induced | pyridimine dimers DNA gyrase- relaxes supercoiling ahead of replication fork Transeri Enzymes ‘An enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription RNA primase- attaches RNA primers to replicating strands DNA polymerase- brings nucleotides and creates daughter strand; also proofreads bases to make sure they are correct. | Exonuclease- finds and removes RNA primers DNA ligase- adds the phosphate backbone; joins 3° end of DNA that replaces primer to rest of leading strands and joins ‘okazaki fragments of lagging strand Primase- synthesizes RNA primer Repressible operon The structural genes are transcribed until they are turned off.ot Te rae necepeans poids hs PO ZY Aah as = tre © repress inact, pero on. When he luce tons idReplication Transcription: A strand of mRNA is synthesized using a specific portion of the cell's DNA as a template. Replication: one parental double stranded DNA molecule is converted into two identical offspring DNA. Each new strand of DNA includes one original strand and one synthesized strand. | | ‘Always happens starting at the original strands 3° end | (attaches a 5') to the original strands 5' end. DNA polymerase can only add new nucleotides to 3° end thus creating the | | leading and lagging strand. The leading strand is the one that | | can be synthesized starting at the original strand’s 3’ end and | continuously made. The lagging strand creates okazaki fragments by starting with an RNA primer, primase, in many | places along the strand. The DNA polymerase extends this | primer creating a longer strand. It then goes back and digests __ | the primer and replaces it with DNA. DNA ligase then joins the | fragments. | | | DNA polymerase proofreads each base pairing and excises | | ones that fit improperly and replace them. | Transcription } Translation | Transcription is the synthesis of a complementary strand of RNA from a DNA template. This strand is typically messenger RNA or mRNA. Transcription begins when RNA polymerase binds to the DNA at a site called a promoter (TATA box). One strand serves as the template, synthesized 5' to 3’ so the original strand was 3' to 5'. RNA synthesis continues until RNA polymerase reaches a site called a terminator. bia | Sense codons- code for amino acids | Nonsense codons- do not code for amino acids. Signal the end | of protein molecule’s synthesis. STOP codon | ‘Synthesis of proteins based on sets of 3 base pairs called codons. Codons call for specific amino acids. Start codon | AUG, which also code for an amino acid, allows the reading to | begin in the right frame. Translation happens in the ribosome | | where tRNA molecules recognize the specific codons and transport required amino acids. Each tRNA has an anticodon sequence that codes for a specific codon's bases. The ribosomes make sure that the right tRNA is binding at the right time t match the mRNA strandStart: AUG STOP: UAG- U Go Away UAA. U are away UAG- U Are Gone DNA is copied by DNA polymerase 5’ to 3. Initiated by an RNA primer. Leading strand is synthesized continuously while the lagging strand is synthesized discontinuously in Okazaki fragments. Lagging strand requires more primers. Primers are removed and Okazaki fragments are joined by a DNA polymerase and DNA ligase.Lactose) Operon —_| Operon- a group of genes that transcribed together and (inducible operon) _| controlled by one promoter. The lac operon consists of three lac structural genes and the adjoining control regions. The lac | operon is an inducible operon meaning it can be tumed off or | ‘on by a gene. In the absence of lactose , the repressor binds to the operator site, thus preventing transcription. If lactose is | present, the repressor binds a metabolite of lactose instead of the operator, and lactose digesting enzymes are transcribed. | This process also depends on the absence of glucose. If | glucose is not available cAMP binds to allosteric site of CAP (catabolic activator protein) causing it to bind with the lac | promoter to make it easier for RNA polymerase to bind to the | promoter acting as an inducer. | | Mutations should be able to differentiate Sense codons code for amino acids Nonsense codons (stop codons) do not. Nonsene, Missense, Nonsense codons- UAA, UAG, UGA signal the end of the | | frame shift, base protein molecule's synthesis before the protein is supposed to | substitution ‘stop being made. Nonsense always makes nonfunctional | proteins | Missense mutation- result in change in amino acid. Can form | functioning proteins or non-functioning proteins. Functioning proteins will not be the ones they are supposed to be. This | type of mutation is responsible for sickle cell anemia. | | Base substitution or point mutation- change in one base | Frameshift mutation- insertion or deletion of one or more | | nucleotide pairs. The shifts the translational reading frame and | almost always result in a long stretch of altered amino acids | | and the production of an inactive protein. | | | Spontaneous Mutation- base shift mutation or frameshift | | mutation cause by occasional mistakes during replication. | | Apparently occur in the absence of any mutation causing | | agents. Mutagens ‘Agents in the environment, such as certain chemicals and radiation, that directly or indirectly bring mutations EX: nitrous acid alters adenine with the result that it pairs with | cytosine rather than thymine (base pair mutagen) | | Ames Test | Positive (direct) selection detects mutant cells because they | Including + and - grow or appear different Selection | Negative (indirect) selection detects mutant cells because they | do not grow. They use replica plating in this in which sterile| velvet is pressed on a master plate then pressed on a plate | with histidine and a plate without histidine. The colony that is | grown on the plate with histidine and not on the plate that is | doesn't have histidine is the colony that they will choose. This test is negative selection because it looks for the colonies that | do not grow when no suspected mutagen is added but do grow | when the suspected mutagen is added. | Ames Test: use bacteria to test for carcinogens (mutagens that ‘cause cancer). It tests the reversions of histidine auxotrophs Salmonella. The mutagen and mutant bacteria are incubated together in rat liver extract because of its high concentration of activation enzymes. If the substance being tested is mutagenic | twill cause the reversion of histidine - bacteria to histidine + bacteria at a rate higher than the spontaneous reversion rate. | | The number of observed revertants observed indicates the degree to which a substance is mutagenic and therefore possibly carcinogenicESSAY — Negative Selection RE: Ames Test Transformation Transduction and Conjuganation Transformation- genes are transferred from one bacterium to another as a “naked” DNA in solution. Conjugation- requires contact between living cells. Transfers genes. Cells must be of opposite “mating type’. Single strand of plasmid DNA is transferred and recipient cell makes complementary strand Transduction- Transfer of DNA from donor to a recipient call by a bacteriophage Chapter 9 Recombinate Cells Gene of interest Know Players: Negative selection Genetic Recombination refers to the exchange between DNA molecules to form new combinations of genes on chromosome Negative Selection plate for Ampicilln - resistance (know this)| Nucleotides DNA Polymase DNA polymerase proofreads new molecules of DNA and Plasmids removes mismatched bases before continuing DNA synthesis. Polymers Enzymes Plasmids are self replicating circular molecules of DNA carrying genes that are not usually essential for the cells | | survival | i Restriction Enzymes | Recognize an AATCC sequence and every time they see this | and functions | they will cut the DNA (AATCC just an example). | -Cut specific sequences of DNA -Destroy bacteriophage DNA in bacterial cells, | -Cannot digest (host) DNA with methylated cytosine | Cleaves the DNA into fragments and creates sticky ends, used to isolate gene of interest Polymerase Chain To make multiple copies of a piece of DNA Reaction enzymatically-makes copies of DNA samples. Used to: | -Clone DNA for recombination, amplify DNA to detectable |Ievels- done at crime scenes so you can do tests to match | | DNA -Sequence DNA Diagnose genetic disease and detect pathogens. | Carry new DNA to the desired cell, Shuttle vectors can exist in | several different species, Plasmids and viruses can be used as vectors. The markers allow us to know what is in the cell and whether the introduction of the gene into the plasmid is successful. | How DNA Plasmid & | cut DNA material | |Introns & Exons | Exons: stretches of DNA that code for protein.Introns: intervening stretches of DNA that do not code for ‘| protein. | To synthesize complementary DNA (cDNA) from an mRNA Reverse | Transcriptase | template. | = a |ESSAY: Blue and | To show that you have the recombinant cell. White screening | - Must not be ampicillin resistant | method - Cannot use beta galactose The sticky ends of the gene of interest will attach to the plasmid | When you do the plate the first thing that can happen is you have the recipient cells that are not recombinant cells they will not grow because it is ampicillin sensitive and they will die. ‘What happens if the recipient cell becomes a recombinant cell | and gets the plasmid but not the recombinant plasmid (gene of | teres) grows because thas te ermplcllin resistant gene: | | but is a blue cell (can break down beta galactose and are | ampicillin resistant). White cells are ampicillin resistant but | | cannot break down beta galactose. | Recipient cell, recombinant cell, and recombinant call that got, | the plasmid. | Vertical- occurs during reproduction, between generation of horizontal gene | cells | transfer | horizontal - transfer of genes between cells of the same. generation Chapter 10 Domains only (No Hierarchy) Not on TEST - Final TEST ONLY! Archaea: , Bacteria, Eukarya | Not on test but maybe for final | Phage Typing Scientific study of serum and other bodily fluids. | | | T | Biochemical Test | Bacteriophage live cells plaques area of colony | | | | | SerologyDNA DNA Generally used to determine the genetic distance between 2 hybridization organisms. Hybridization or binding of 70% or more bases AKA nucleic acid indicates 2 organisms belong to the same species. hybridization TEST: Gram + Rod shape with chains Classify Bacteria 1) Gram stain, 2) endospore, 3) shape, 4) aerobic or what we use anaerobic. Gram + rod have endospore - run endospore stain Streptococcus How is it possible to streptococcus to live in the presence of ‘oxygen with being catalase +? Have Chapter 11 Streptomyces Why it is important? Medical: They Produce most of commercial antibiotics. Mycobacterium TB - acid fast, Gram Positive Pseudomonas Where do you see pseudomonas? Catheters (UTI), Respiratory Tract, Wounds. (Multi-organism)Ea I |Rhizobium (Gram | Agriculture - Bacteria that fix nitrogen to for plants. | | Negative Bacteria) Clostridium Gram positive, endospore formation, anaerobic Yersinia Gram negative, small rod, found in europe, caused 1500's Black Plague. ae ee eee ea a | Examples of Gram negative, facultative, e-coli, enterobacter, salmonella, Enterobacteriales _ shigella | Bacillus | Gram positive, rod shape, aerobic | Ifyou havea gram | Endospore test because gram positive rods can form Positive rod, what _| endospores. (bacillus and clostridium) test would you run | next? | Normally a Escherichia coli commensal in the human intestine, this | bacterium became pathogenic after acquiring a toxic gene from a Shigella bacterium. Lac operon ‘An operon required for the transport and metabolism of lactose in E coli and many | other bacteria. The operon consists of the promotor and the operator sites and structural | | genes that code for the proteins. The operon is regulated by the product of the regulatory | gene. The repressor protein binds with the operator, preventing transcription from the ‘operon. When the inducer allolactose binds to the repressor protein, the inactivated | repressor can no longer block transcription. The structural genes are transcribed, | ultimately resulting in the production of the enzymes needed for lactose catabolism. This process also depends on the absence of glucose. If glucose is not available cAMP binds to allosteric site of CAP (catabolic activator protein) causing it to bind with the lac | promoter to make it easier for RNA polymerase to bind to the promoter acting as an inducer. Lo| eubacteria and archaea | | A:Archaea- cell wall- no peptidoglycan Eubacteria- peptidoglycan Archeae- not sensitive to antibiotics E- sensitive First amino acid in all protein for A- methionine and E- formyimethionine | Viruses A: 1. viruses are acellular particles- they are not made of living cells- and consist of a central core of DNA or RNA surrounded by a protein coat 2. They lack the properties of living things. | -Viruses cannot reproduce independently and require a host cell to replicate ~The have no energy metabolism | “They do not grow | They produce no waste products | -They do not respond to stimuli What would UV rai ion Target A CTTTGA will be most likely because UV light is known to break DNA and cause | Thymine- Thymine dimers. All bacteria are not killed because some have capsules and | endospores for protection while other have the ability to fix the dimers created to restore their DNA through light repair or nucleotide excisions. | Transformation Pr | ple (a) Living encapsulated bacteria caused disease and death when injected into a mouse. (b) Living nonencapsulated bacteria are readily destroyed by the phagocytic defenses of the host of the host, so the mouse remained healthy after injection. | (c) After being killed by heat, encapsulated bacteria lost the ability to cause disease. (d) However, the combination of living nonencapsulated bacteria and heat-killed | encapsulated bacteria (neither of which alone causes disease) did cause disease. | Somehow, the live nonencapsulated bacteria were transformed by the dead encapsulated bacteria so that they acquired the ability to form capsules and therefore cause disease, Subsequent experiments proved the transforming factor to be DNA. | L os | ‘Blue-White Screeningplasmid has the genes for lactose hydrolysis (the JacZ gene encodes the enzyme beta-galactosidase) and ampicillin resistance. | 2. Foreign DNA will insert into the /acZ gene. The bacterium receiving the plasmid | vector will not produce the enzyme beta-galactosidase if foreign DNA has been inserted into the plasmid. 3. The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant. | 4. All treated bacteria are spread on a nutrient agar plate containing ampicillin and a | | beta-galactosidase substrate and incubated. The beta-galactosidase substrate is called | | X-gal. | 5. Only bacteria that picked up the plasmid will grow in the presence of ampicillin. | | | Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indigo | | tums the colonies blue. Bacteria that cannot hydrolyze X-gal produce white colonies. | ‘A: 1. Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The | AMES TEST | A:Many known mutagens have been found to be carcinogens, substances that cause | cancer in animals, including humans. The Ames test is a faster less expensive procedure for the preliminary screening of potential carcinogens. It uses bacteria as carcinogen | | indicators. It is based on the observation that exposure of mutant bacteria to mutagenic | substances may cause new mutations that reverse the effect of the original mutation.