BIOL 261: Study Guides for Exam 2 (Chs. 7-11)
BIOL 261: Study Guides for Exam 2 (Chs. 7-11) BIOL 261
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This 38 page Study Guide was uploaded by Alexis Ward on Saturday October 8, 2016. The Study Guide belongs to BIOL 261 at University of Louisiana at Lafayette taught by Professor P. Antley in Fall 2016. Since its upload, it has received 9 views. For similar materials see General Microbiology in Microbiology at University of Louisiana at Lafayette.
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Date Created: 10/08/16
Chapter 8 – “ Viruses and Virology” Handout Vocabulary • Bacteriophage- A virus that infects prokaryotic cells. • Capsid- The protein shell that surrounds the genome of a virus. • Capsomere- The subunit of the virus capsid. • Early protein- A protein synthesized soon after virus infection and before replication of the virus genome. • Host cell- An organism or cell type capable of supporting the growth of a virus or other parasite. • Icosahedron- A geometrical shape occurring in many virus particles, with 20 triangular faces and 12 corners. • Late protein- A protein synthesized later in virus infection after replication of the virus genome. • Lysogen- A prokaryote containing a prophage. See also temperate virus. • Lysogeny- A state following virus infection in which the viral genome is replicated as a provirus along with the genome of the host. • Lytic pathway- A series of steps after virus infection that lead to virus replication and the destruction (lysis) of the host cell. • Nucleocapsid- The complete complex of nucleic acid and protein packaged in a virus particle. • Plaque- A zone of lysis or growth inhibition caused by virus infection on a lawn of host cells. • Prophage- The state of the genome of a temperate virus when it is replicating in synchrony with that of the prokaryotic host, typically integrated into the host genome. See provirus. • Provirus- The genome of a temperate or latent animal virus when it is replicating in step with the host chromosome. • Retrovirus- A virus whose RNA genome has a DNA intermediate as part of its replication cycle. • Reverse transcription- The process of copying information found in RNA into DNA. • Temperate virus- A virus whose genome is able to replicate along with that of its host without causing cell death in a state called lysogeny. • Transformation- (1) The transfer of genetic information via free DNA. (2) A process, sometimes initiated by infection with certain viruses, whereby a normal animal cell becomes a cancer cell. • Virion- A virus particle; the virus nucleic acid surrounded by a protein coat and in some cases other material. • Virulent virus- A virus that lyses or in some other way kills the host cell after infection; a nontemperate virus. • Virus- A genetic element that contains either RNA or DNA and that replicates only in host cells; has an extracellular form. Review Questions 1) What are the basic components of a virus? - Capsid- The protein shell that surrounds the genome of a virus. Capsomere- The subunit of the virus capsid. Nucleocapsid- The complete complex of nucleic acid and protein packaged in a virus particle. Enveloped virus- virus that contains additional layers around the nucleocapsid - Naked virus: nucleic acid, capsid Enveloped virus: nucleic acid, capsid, envelope - The protein coat that surrounds the viral nucleic acid is called a capsid and the individual subunits that make up the capsid are called capsomers. When a virus buds out of its host cell, it takes with it part of the cytoplasmic membrane. This membrane, once it is on the virus, is called an envelope. If the virus does not have an envelope it is said to be a naked virus. (Exs: HIV and influenza are examples of enveloped viruses.) 2) What types and arrangements of nucleic acids are found in viruses? - Nucleic acids are made up of either DNA or RNA and are either single stranded (ss) or double stranded (ds). Some are circular, but most are linear. - The key words when it comes to talking about viral genomes is either and or!! Viruses have either DNA or RNA. Because they don’t have both and are not able to reproduce independently they are not cells. 3) What is a host? Bacteriophage? - Host cell- An organism or cell type capable of supporting the growth of a virus or other parasite. - Bacteriophage- A virus that infects prokaryotic cells. (Exs of phages: Lambda and T4.) 4) What units are used to measure viruses? - Viruses are measure in nanometers (nm), because they are so small. Smaller than bacteria which is measured in microns (um). 5) What is a capsid, capsomere, envelope, and naked virus? - Capsid- The protein shell that surrounds the genome of a virus. Capsomere- The subunit of the virus capsid. Naked virus: nucleic acid, capsid Enveloped virus: nucleic acid, capsid, envelope (a lipoprotein membrane surrounding the virion.) 6) What are capsids made of? Viral envelopes? - Capsids are made of capsomeres. - Viral envelopes come from the host cell cytoplasmic membrane, so it is made of lipid bilayer; it may contain proteins, called spikes, as in the case of the influenza virus. 7) Describe a complex virus and give an example. - Complex Viruses: virions composed of several parts, each with separate shapes and symmetries. - Bacterial viruses contain complicated structures: icosahedral heads (geometrical shape occurring in many virus particles, with 20 triangular faces and 12 corners) and helical tails. 8) How is T4 and Lambda similar and different? - Lambda and T4- both complex structures and are DNA viruses (both phage viruses.) - Lambda- lysogenic (temperate) phage = have a prophage stage where the viral DNA goes into the host chromosome. (Electron micrograph by negative staining of phage lambda virions. The head of each virion is about 65 nm in diameter and contains linear dsDNA). Temperate virus- A virus whose genome is able to replicate along with that of its host without causing cell death in a state called lysogeny; this is due to repressor proteins. - T4- lytic phage = lyse the host. Virulent virus- A virus that lyses or in some other way kills the host cell after infection; a nontemperate virus. 9) How is the penetration step in viral replication different in phages and animal viruses? What else would be different in their replication? - Penetration step different: the capsid of phages is left outside the host following penetration & with animal viruses, the entire virus is taken into the host and must be uncoated to release the nucleic acid. Remember that animal cells are eukaryotic and thus have a nucleus so other parts of the viral replication process will be different from phages. - Replication step different: (in animal viruses) there is receptor-mediated endocytosis where enveloped viruses adsorb to the host cell with their protein spikes and the viruses are taken into the cell in a process termed endocytosis. In this process, the host cell plasma membrane surrounds the whole virion and forms a vesicle. Then, the envelope of the virion fuses with the plasma membrane of the vesicle. The nucleocapsid is then released into the host's cytoplasm. Animal viruses bud out of the host cell so they will have an envelope which comes from the cytoplasmic membrane of the host cell. 10)What is unique about retroviruses? Name a retrovirus? Describe how retroviruses reproduce. - Retrovirus- A virus whose RNA genome has a DNA intermediate as part of its replication cycle. Retro means “backwards”; are RNA viruses that contain the enzyme reverse transcriptase which copies the single stranded RNA into double stranded DNA which then integrates into the host chromosome and becomes a provirus. (Recall that transcription is DNA to RNA.) - (Exs: HIV was the first virus to be linked to cancer. It is a retrovirus that infects T- Helper lymphocytes. As the virus buds out of the T-cells, the lymphocytes eventually die off and the person’s T-cell count drops to below 200 and they then have full blown AIDS, which makes them immunocompromised and thus are susceptible to opportunistic infections.) - Retroviruses reproduce: Entrance into the cell, Removal of virion envelope at the membrane, Reverse transcription of one of the two RNA genomes, Integration of retroviral DNA into host genome, Transcription of retroviral DNA, Assembly and packaging of genomic RNA, and Budding of enveloped virions/Release from cell. 11)How is a lytic virus different from a latent virus? What other terms are used to describe latent viruses? - Lytic virus- virus that goes into the cell to immediately lyse it (explodes it) to spread. - Latent virus- viruses that enter a dormant state and not replicate until some trigger causes them to activate and become either persistent or lytic; also called Proviruses. 12)How is prophage different from a provirus? - Prophages and Proviruses are similar in that they both insert their genetic material into the nucleus of the host cell. - In Prophage (lytic) reproduction (in bacteria only) the genetic material leaves the nucleus as the cell is destroyed. - In Proviral (latent) reproduction, the genetic material will permanently remain in the nucleus. 13)What keeps temperate phages latent? - They may undergo infections in which they may not produce virions for a long period of time. 14)When an animal cell gets infected with a virus what are the possible consequences? How can a virus cause cancer? - Persistent infections: release of virions from host cell does not result in cell lysis; infected cell remains alive and continues to produce virus. - Latent infections: delay between infection by the virus and lytic events. Transformation: conversion of normal cell into tumor cell. Cell fusion: two or more cells become one cell with many nuclei. Learning Objectives Identify the functions of the following enzymes…lysozyme, polymerase, neuraminidase Identify the host for T4, lambda, HIV, Describe the possible results of viral infections. Describe the steps in both latent and lytic viral infections in bacteria and animals. Compare and contrast a viral growth curve to a bacterial growth curve. Describe the infection and reproduction of a retrovirus like HIV MM Qs and As: Viral replication is - independent of the host cell's DNA but dependent on the host cell's enzymes and metabolism. Reverse transcriptase is a(n) - RNA-dependent DNA polymerase. Which of the following enzymes would you expect to find in the virion of a retrovirus, but NOT in a bacteriophage? - Reverse transcriptase The term "phage" is generally reserved for the viruses that infect - Bacteria For bacteriophages and animal viruses ________ is the step in the viral life cycle that determines host cell or tissue specificity. - Attachment What are the consequences of a viral infection of an animal cell? - Outcomes vary from rapid lysis to persistent infections, latent infections, or cancer. Lysogeny is unique to bacteriophages; similar relationships have not been found among the animal viruses. - False Viral size is generally measured in - Nanometers Enveloped viral membranes are generally ________ with associated virus-specific ________. - lipid bilayers / glycoproteins When packaged in the virion, the complete complex of nucleic acid and protein is known as the virus - Nucleocapsid Which of the following are the hosts for most enveloped viruses? - Animals Regarding the viral membrane of an enveloped virus, the lipids are derived from the ________, and the proteins are encoded by ________. - Host’s cell membrane / viral genes Some viruses possess icosahedral heads and helical tails. - True Figure 8.6 (the replication cycle of a bacterial virus): Attachment, Penetration of viral nucleic acid, Synthesis of viral nucleic acid and proteins, Assembly and packaging of new viruses, & Cell lysis and release of new virions. Figure 8.7 (one-step growth curve of virus replication) Viral replication occurs - Intracellularly When a virus enters a host cell in which it can replicate, the process is called a(n) - Infection The growth of viruses in a culture is described as a one-step growth curve, because - virion numbers show no increase during intracellular replication and can only be counted after the virions burst from the host cell. Viruses have both an intracellular and an extracellular form. - True Viruses can redirect host metabolic functions. - True In the first few minutes after host cell infection, the virus undergoes an eclipse. - True Penetration requires that the entire virus is inserted within the host. - False A lytic infection results in death of the host cell. - True The latent phase in the viral growth curve and the lag phase of the bacterial growth curve are equivalent and represent the time it takes for the virus or bacterium to adapt to the culture conditions and begin growing. - False Figure 8.8 (quantification of bacterial virus by plaque assay): the cell-phage mixture is poured onto a solidified nutrient agar plate, the mixture is left to solidify, and the incubation allows for bacterial growth and phage replication. Viruses infecting ________ are typically the easiest to grow in the laboratory. - Prokaryotes The use of ________ is the easiest and most effective way of studying many animal and plant viruses. - Tissue or cell culture When solutions of host cells and infectious virions are mixed and spread on an agar plate, ________ form where viruses lyse the host cells. - Plaques The concentration of infectious plaque forming units (pfu) per volume of fluid is known as the - Titer Cellular receptors may be composed of - combinations of proteins, carbohydrates, and/or lipids. Bacteriophage have a ________ complex structure than animal viruses, because ________. - more / the bacteriophage must penetrate the peptidoglycan cell wall Virions infecting some bacteria possess the enzyme ________ that makes a small hole in the bacterial cell wall, allowing the viral nucleic acid to enter. - Lysozyme Figure 8.13 (time course of events in phage T4 infection) Part A: How are viruses different from cells? - They require a host in order to reproduce. Part B: What is the function of the structural elements of a virus? - To package and protect the viral genome. Part A: In which stage is the viral DNA introduced into the cell? - Penetration Part B: In which stage does formation of mature viruses occur? - Assembly Part C: The host DNA is usually degraded during which stage? - Biosynthesis Part D: What would be the fate of a lytic bacteriophage if the host cell died prior to the assembly stage? - The virus would not be able to infect new hosts Figure 8.15 (consequences of infection by a temperate bacteriophage) Part A: Lysogenic viral DNA integrating into the host genome is referred to as - A prophage Part B: Which of the following events might trigger induction of a temperate bacteriophage? - Exposure to UV light Part C: How is the lytic cycle different from the lysogenic cycle with respect to the infected host cell? - The host cell dies during the lytic stage Part D: What is the fate of the prophage during the lysogenic stage? - It is copied every time the host DNA replicates. A virus that kills its host is said to be - Lytic or virulent A prophage replicates - along with its host while the lytic genes are not expressed. Temperate viruses can enter into either a lytic or lysogenic cycle. - True Figure 8.24 (replication of a retrovirus): Entry and uncoating of the retrovirus, Reverse transcriptase activity (two steps), Viral DNA enters the nucleus and intergrates into the host genome, Transcription by host RNA polymerase forms viral mRNA and genome copies, & Translation of mRNA forms viral proteins (new nucleocapsids assembled and released by budding) Chapter 9 – Study Guide Vocabulary Antigenic Shift- In influenza virus, major changes in viral proteins (antigens) due to gene reassortment. Hepadnavirus- A virus whose DNA genome replicates by way of an RNA intermediate. Negative strand- A nucleic acid strand that has the opposite sense to (is complementary to) the mRNA; A virus with a single-stranded genome that has the opposite sense to (is complementary to) the viral mRNA. Positive strand- A nucleic acid strand that has the same sense as the mRNA; A virus with a single-stranded genome that has the same complementarity as the viral mRNA. Prion- An infectious protein whose extracellular form contains no nucleic acid. Retrovirus- A virus whose RNA genome has a DNA intermediate as part of its replication cycle. Reverse Transcription- The process of copying information found in RNA into DNA. Viroid- Small, circular, single-stranded RNA that causes certain plant diseases. Study Questions 1) What type of nucleic acids are found in viruses? - Viruses have either DNA (replicate in nucleus) or RNA (replicate in cytoplasm) genomes. Some are circular, but most are linear. 2) Which organisms have the largest genome? - DNA virus: (Smallest) Circovirus: 1.75 kilobase (kb) single strand (Largest) Megavirus: 1.25 megabase (mb) pairs - RNA viruses: (Smallest) MS2: 3.5 kilobase (kb) (Largest) Coronavirus: 32 kilobase (kb) 3) What is unique about the Pox virus? Adenoviruses? - Pox virus: among the most complex and largest animal viruses known; causes small pox; DNA replicates in the cytoplasm (abnormal; normally replicates in the nucleus) - Adenovirus: major group of icosahedral, linear, double-stranded DNA viruses; causes mild respiratory infections in humans; DNA replicates in the nucleus which requires protein primers and avoids synthesis of a lagging strand. 4) Name two animal viruses that can induce cancer. How is that possible? - Polyomavirus SV40: non-enveloped virion with an icosahedral head; no enzymes in the virion; replicates in host nucleus; DNA is circular. - Herpesviruses: able to remain latent for extended periods of time; infection follows attachment of virions to specific cell receptors; (Ex: Epstein–Barr virus- associated with mono); three classes of mRNA are produced: Immediate early: encodes five regulatory proteins Delayed early: encodes DNA replication proteins Late: encodes structural proteins of the virus particle (Both are DNA viruses) - Can induce cancer, because the viral DNA goes into the host chromosome (is now called a provirus *in bacteria called prophages*), causes a mutation (a change in the nucleotide sequence) that causes the cells to go from normal cells (with contact inhibition) to tumors (having lost the contact inhibition.) *contact inhibition: a regulatory mechanism that functions to keep cells growing into a layer one cell thick (a monolayer) until all of a substrate it is on is filled. 5) Which virus is associated with mononucleosis? - The Herpesvirus is associated with mono. 6) What is a positive strand RNA virus? How is it replicated? - Positive strand- A nucleic acid strand that has the same sense as the mRNA; A virus with a single-stranded genome that has the same complementarity as the viral mRNA. - Replication of positive-strand RNA viruses requires a negative-strand RNA intermediate from which new positive strands are synthesized. 7) What is a negative strand RNA virus? How is it replicated? - Negative strand- A nucleic acid strand that has the opposite sense to (is complementary to) the mRNA; A virus with a single-stranded genome that has the opposite sense to (is complementary to) the viral mRNA. - Replication of negative-strand RNA viruses into mRNA by an enzyme present in the virion. 8) What type of virus is poliovirus? - Poliovirus: a small virus where viral RNA is translated directly, producing a single long, giant protein (polyprotein) that undergoes self-cleavage to generate ~20 smaller proteins necessary for nucleic acid replication and virus assembly. Host RNA and protein synthesis are inhibited when poliovirus replication begins *Is a + Strand RNA virus* 9) What disease is associated with coronaviruses? - Coronavirus: a larger virus that causes respiratory infections, including SARS, in humans and other animals. *Is a + Strand RNA virus* - The Coronavirus causes SARS- sudden acute respiratory syndrome = fever. 10)What type of virus are rhabdoviruses and what disease are they associated with? What shape capsid do they have? - Rhabdoviruses: include viruses that cause rabies in animals and humans and vesicular stomatitis in cattle, pigs, & horses. Have enveloped viruses & the virion (are enveloped, contain club-shaped glycoprotein spikes on their surfaces & produces monocistronic mRNA) is bullet-shaped. RNA of rhabdoviruses is transcribed in the host cytoplasm into two distinct classes: Series of mRNAs encoding the structural genes of the virus. Positive-strand RNA that is a copy of the complete viral genome. *Is a – Strand RNA virus* - The Rhabdovirus has a bullet-shaped capsid. 11)What type of virus is influenza? - Influenza: an enveloped, pleomorphic virus with a segmented genome; surface proteins interact with the host cell surface: Hemagglutinin causes clumping of red blood cells. Neuraminidase breaks down the sialic acid component of the host cytoplasmic membrane. *Is a – Strand RNA virus* 12)What is the function of hemagglutinin and neuraminidase? Where are they found? - (H1) Hemagglutinin: causes clumping of red blood cells. - (N1) Neuraminidase: breaks down the sialic acid component of the host cytoplasmic membrane. - These surface proteins interact with the host cell surface of influenza. 13)How is antigenic shift different from antigenic drift? - Antigenic shift: where portions of the RNA genome from two genetically distinct strains of virus infecting the same cell are re-assorted; generates virions that express a unique set of surface proteins; results when two different flu strains combine and infect the same cell. - Antigenic drift: where the structure of neuraminidase and hemagglutinin proteins are subtly altered; changes to the flu virus that happen slowly over time. This causes the changes to the seasonal flu that require us to get vaccinated against the flu each year. - These are processes that help influenza elude the host’s immune system. 14)What are reoviruses associated with? - Reoviruses: are non-enveloped nucleocapsid with a double shell of icosahedral symmetry; virions contain virus-encoded enzymes necessary to synthesize mRNA and the new RNA genomes. Genome is segmented into 10–12 molecules of linear double-stranded RNA and replication occurs exclusively in host cytoplasm within the nucleocapsid. Respiratory, enteric, orphan viruses! *Is a dsRNA virus* - Reoviruses are not associated with any specific thing. 15)What type of virus are retroviruses and how do they replicate? Give an example of a retrovirus. - Retroviruses: are enveloped virions that contain two copies of the RNA genome; virion contains several enzymes. Includes reverse transcriptase used to make the DNA copy of the genome. All retroviruses have the three genes: gag encodes several small viral structural proteins. pol is translated into a large polyprotein. The env product is processed into two distinct envelope proteins. *Is an RNA virus* - (Ex: HIV. Uses reverse transcriptase that take ssRNA, make dsDNA which goes into the host chromosome (where it is called a “provirus”) and is then “latent”. The host of HIV is T-Helper cells (a type of lymphocyte). When they do become active and make new viruses, they leave the host by budding out and become enveloped (cell or cytoplasmic membrane of the host). Eventually, T- cell will die and the T-cell count goes from 600 (normal) to <200 and have full blown AIDS. When the count goes to 200, person has acquired immunodeficiency and become susceptible to other infections that could cause death. - Retroviruses (RNA viruses) and hepadnaviruses (DNA viruses) use reverse transcriptase for replication. 16)What is the host for hepadnaviruses? - Hepadnaviruses: virions are small, irregular-shaped particles; Include hepatitis B; Viral replication occurs through an RNA intermediate; have unusual genomes: Tiny. Only partially double-stranded. *Is a DNA virus* - Humans, apes, and birds serve as hosts. 17)What is a viroid? What does it infect? What is unique about it? - Viroids: infectious RNA molecules that lack a protein coat; smallest known pathogens (246–399 bp) that cause a number of important plant diseases; have small, circular, ssRNA molecules (single stranded RNA that’s rolled back onto itself) and do not encode proteins; completely dependent on host-encoded enzymes. - Viroids are infectious RNA molecules that lack a protein coat (capsid) and infect plants. 18)What is a prion? How does it cause disease? - Prions: infectious RNA proteins whose extracellular form contains no nucleic acid and are known to cause disease in animals (transmissible spongiform encephalopathies); Host cell contains gene (PrnP) that encodes native form of prion protein that is found in healthy animal. - Prion misfolding results in neurological symptoms of disease (e.g., resistance to proteases, insolubility, and aggregation.) 19)What are some prion diseases? - Prion diseases are Mad Cow Disease, kuru (associated with cannibals), Creurfeldt Jakob disease (most common.) Learning Objective To be able to compare and contrast the basic differences between reproduction of DNA vs RNA viruses MM Qs and As: Which type of viruses generally has the smallest genome? - RNA viruses Which type of viruses can be directly used for translation? - + ssRNA (positive single stranded RNA) The Baltimore classification scheme is a useful way to categorize viruses based on their host infectivity. - False Herpesviruses can cause all of the following diseases in humans EXCEPT - spongiform encephalopathy. What is the purpose of synthesizing a negative strand RNA in positive stranded ssRNA viruses? - to serve as the complementary template sequence in genome amplification of the positive strand Figure 9.19b: Influenza virus. A drug designed to inhibit reverse transcriptase activity would target - hepadnaviruses and retroviruses. A bacteriophage that lacks its proteinaceous capsid structure is also called a viroid. - False Part A: The normal function of the PrP protein in mammals is believed to be: - assisting in normal synaptic development and function. Part B: How do normal prion proteins (PrP) differ from the infectious prion proteins? - Normal PrP have alpha-helices; infectious PrP have beta-pleated sheets. Part C: How does the number of infectious prions increase? - Prions transform normal proteins into the misfolded beta-pleated sheet configuration; therefore, prions multiply by conversion. Part D: Why are the beta-pleated multimers of PrP potentially pathogenic? - The multimers are more stable and resistant to protease. Part A: Which of the following prion diseases is found in deer and elk? - Chronic wasting disease Part B: Which of the following prion diseases was also known as laughing disease? - Kuru Part C: Which of the following conditions in humans is linked to bovine spongiform encephalopathy? - Variant Creutzfeldt-Jakob disease Part D: What part of the nervous system is most affected by fatal familial insomnia? - Thalamus Part E: Where does the name “scrapie” come from? - The prion disorder causes infected sheep to scrape against objects until their skin is raw. Part A: From which phrase is the term “prions” derived? - Proteinaceous infectious particles Part B: In what year did Stanley Prusiner discover prions? - 1982 Part C: Which disease did Stanley Prusiner first identify as being caused by prions? - Scrapie Part D: How are prions different from other infectious agents? - They lack nucleic acid Chapter 10 – Study Guide Vocabulary Auxotroph- An organism that has developed a nutritional requirement through mutation. Contrast with prototroph. Competence- The ability to take up DNA and become genetically transformed. Conjugation- The transfer of genes from one prokaryotic cell to another by a mechanism involving cell-to-cell contact. Electroporation- The use of an electric pulse to enable cells to take up DNA. Genotype- The complete genetic makeup of an organism; the complete description of a cell's genetic information. Compare with phenotype. Frame Shift (mutation)- A mutation in which insertion or deletion of nucelotides changes the groups of three bases in which the genetic code is read within an mRNA, usually resulting in a faulty product. Hfr cell- A cell with the F plasmid integrated into the chromosome. Induced mutation- A mutation caused by external agents such as mutagenic chemicals or radiation. Insertion sequence- The simplest type of transposable element, which carries only genes involved in transposition. Missense mutation- A mutation in which a single codon is altered so that one amino acid in a protein is replaced with a different amino acid. Mutagen- An agent that induces mutation, such as radiation or certain chemicals. Mutant- An organism whose genome carries a mutation. Mutation (insertion, deletion, substitution)- An inheritable change in the base sequence of the genome of an organism. Nonsense mutation- A mutation in which the codon for an amino acid is changed to a stop codon. Phenotype- The observable characteristics of an organism, such as color, motility, or morphology. Compare with genotype. Point mutation- A mutation that involves a single base pair. Recombination- Resorting or rearrangement of DNA fragments resulting in a new sequence combination. Reversion- An alteration in DNA that reverses the effects of a prior mutation. Rolling circle replication- A mechanism, used by some plasmids and viruses, of replicating circular DNA, starting by nicking and unrolling one strand. For a double- stranded genome, the unrolled strand is used as a template for DNA synthesis; for a single-stranded genome, the other, still circular strand is used as the template for DNA synthesis. Screening- A procedure that permits the identification of organisms by phenotype or genotype, but does not inhibit or enhance the growth of particular phenotypes or genotypes. Selection- Placing organisms under conditions that favor or inhibit the growth of those with a particular phenotype or genotype. Silent mutation- A change in DNA sequence that has no effect on the phenotype. Spontaneous mutation- A mutation that occurs "naturally" without the help of mutagenic chemicals or radiation. Transduction- The transfer of host genes from one cell to another by a virus. Transfection- The transformation of a prokaryotic cell by DNA or RNA from a virus. Used also to describe the process of genetic transformation in eukaryotic cells. Transformation- (1) The transfer of genetic information via free DNA. (2) A process, sometimes initiated by infection with certain viruses, whereby a normal animal cell becomes a cancer cell. Transposon- A type of transposable element that carries other genes in addition to those involved in transposition; often these genes confer selectable phenotypes such as antibiotic resistance. Wild-type strain- A strain of microorganism isolated from nature or one used as a parent in a genetics investigation. The usual or native form of a gene or organism. Study Questions 1) What is a mutation? What causes mutations? What is a wild-type strain? - Mutation- A heritable change in DNA sequence that can lead to a change in phenotype (observable properties of an organism); An inheritable change in the base sequence of the genome of an organism. Mutant- An organism who’s genome carries a strain of any cell or virus differing from parental strain in genotype (nucleotide sequence of genome). - Mutagen- An agent that induces/causes mutation, such as radiation or certain chemicals. - Wild-type strain- Typically refers to strain isolated from nature or one used as a parent in a genetics investigation; the usual or native form of a gene or organism. 2) How is a selectable mutation different from a nonselectable mutation? - Selectable mutations: those that give the mutant a growth advantage under certain conditions & are useful in genetic research. (Ex: If you are looking for a bacteria that is resistant to ampicillin, then you can grow the bacteria on media containing ampicillin. Anything that grows on Amp media is resistant to ampicillin.) - Non-selectable mutations: those that usually have neither an advantage nor a disadvantage over the parent (just happen and are more difficult to find) & detection of such mutations requires examining a large number of colonies and looking for differences (screening). (Ex: There is no media that you could grow your cells on that selects for colonies that are a certain color. The process of looking for the colonies that have a specific color, or phenotype, that is non-selectable is called screening. A type of screening is replica plating.) 3) Explain how replica plating works. What is an auxotrophic mutant? - Replica plating: is useful for identification of cells with a nutritional requirement for growth (auxotroph). This technique is used to identify/isolate auxotrophic mutants which are mutants that have a nutritional growth requirement that must be given to them or else they will not grow. - Process-- You press the master plate (the plate of growth on the complete medium) to the velveteen (which is sterilized), the velveteen now has the imprint of all of the colonies, you then transfer the imprint of all colonies to fresh media (a complete and minimal medias), incubate, (on the complete media) all colonies grow & (on the minimal media) the mutants do not grow. - Auxotrophic mutant- An organism that has developed a nutritional requirement through mutation. Contrast with prototroph. 4) How is an induced mutation, spontaneous mutation and a point mutation similar and different? - Induced mutation- A mutation caused by external agents such as mutagenic chemicals or radiation; ones in which you intentionally expose a cell to a mutagen. - Spontaneous mutation- A mutation that occurs "naturally" without the help of mutagenic chemicals or radiation/external intervention; mutations that “just happen.” - Point mutations- Mutations that change only one base pair that can lead to single amino acid change in a protein, an incomplete protein, or no change at all; only one base changes either through deletion, substitution, or addition – recall, that one base can change the reading frame and thus cause a change in the protein coded for by the gene. 5) What does a point mutation cause? What are the possible ultimate results of a point mutation? - Point mutations cause mutations that change only one base pair. - Results of a point mutation: a single amino acid change in a protein, an incomplete protein, or no change at all. 6) What is a silent, missense and nonsense mutation? Frameshift mutation? Reversion? - Silent mutation- A change in DNA sequence that has no effect on the phenotype. - Missense mutation- A mutation in which a single codon is altered so that one amino acid in a protein is replaced with a different amino acid; polypeptide is altered. - Nonsense mutation- A mutation in which the codon for an amino acid is changed to a stop codon; polypeptide is incomplete. - Frame Shift (mutation)- A mutation in which insertion or deletion of nucelotides changes the groups of three bases in which the genetic code is read within an mRNA, usually resulting in a faulty product. - Reversion- An alteration in DNA that reverses the effects of a prior mutation; mutant goes back to its wild type. Two types of revertants: Same-site revertant: mutation is at the same site as original mutation Second-site revertant: mutation is at a different site in the DNA suppressor mutation that compensates for the effect of the original mutation 7) What are some examples of mutagens? - Mutagen- An agent that induces mutation, such as radiation or certain chemicals. - Several classes of chemical mutagens exist: Nucleotide base analogs: resemble nucleotides (Ex: AZT used to treat HIV infections; Acylovir used to treat Herpes infections; causes not to make nucleic acid its supposed to make and cause it to die; used in treatment.) Chemical mutagens that cause chemical modifications (Ex: alkylating agents like nitrosoguanidine, nitrous acid) Chemical mutagens that cause frameshift mutations (Ex: intercalating agents like acridines, Ethidium Bromide, Agent Orange) 8) What is a nucleotide base analog? What does it do to a cell? - Nucleotide base analogs are a type of chemical mutagen that resemble nucleotides. Analogs are chemicals or structures that resemble another structure. If the cell or virus gets the nucleotide then all is good; however, if it gets the base analog then it will not be able to base pair correctly and a mutation will occur. This principle is used in treatment of some of the viral diseases i.e. AZT. 9) How is UV light different from gamma rays? What do they do to the cell? How is the damage repaired? What happens if the cell can’t repair the damage to the DNA? - Two main categories of mutagenic electromagnetic radiation: Non-ionizing (i.e., UV radiation): Purines and pyrimidines strongly absorb UV. Pyrimidine dimer is one effect of UV radiation. (Ex: UV light causes T-T dimers to form in cells which, if not repaired, can lead to mutations that may result in skin cancer.) Ionizing (i.e., X-rays, cosmic rays, and gamma rays): Ionize water and produce free radicals which damage macromolecules in the cell. (Ionizing radiation goes all the way through a person and it is more deadly, because it can cause actual breaks in the DNA which will lead to cell death.) - When DNA damage is large scale, the cell may use a different type of repair system (Ex: damage interferes with DNA replication) Mechanism called the SOS regulatory system: This system is more error prone, but allows replication to proceed and the cell to replicate. 10)Explain how the Ames test works and what it is used for. - The Ames test: makes practical use of bacterial mutations to detect for potentially hazardous chemicals/carcinogens; looks for an increase in mutation of bacteria in the presence of suspected mutagen. A wide variety of chemicals have been screened for toxicity and carcinogenicity. - It is done with and auxotrophic mutant and if the chemical causes a back mutation then it is a potential carcinogen. 11)What is transformation? Explain how Griffith’s transformation experiment worked. - Transformation- (1) The transfer of genetic information via free DNA. (2) A process, sometimes initiated by infection with certain viruses, whereby a normal animal cell becomes a cancer cell; the uptake of free DNA. It is a technique used to clone genes into bacteria. Discovered by Fredrick Griffith in the late 1920s. He worked with Streptococcus pneumoniae. This process set the stage for the discovery of DNA. - Fredrick Griffith discovered this processes while working with the bacteria Streptococcus pneumonococcus, which has a capsule when virulent -- causes pneumonia and doesn’t have a capsule when non-pathogenic. The gene for the capsule is carried on a plasmid. - Live smooth (S) cells contain a capsule and kill mice, because immune cells cannot kill the encapsulated bacteria; the cells proliferate in the lung and cause a fatal pneumonia. Rough (R) cells have no capsule and are not pathogenic. A combination of live (R) and dead (S) cells kill mice, and live (S) cells can be isolated from the animals. DNA carrying genes for capsule production is released from dead (S) cells and taken up by (R) cells, thus transforming them into live (S) cells. * The gene for the capsule of Streptococcus pneumoniae are carried on the plasmid of the smooth cells. When these cells die, they lyse and release the plasmid. The rough cells pick up the plasmid through transformation and become encapsulated.* 12)What does it mean to say a cell is competent? - Competent: cells capable of taking up DNA and being genetically transformed; In naturally transformable bacteria, competence is regulated. In other strains, specific procedures are necessary to make cells competent. Electricity can be used to force cells to take up DNA (electroporation) (Ex: Bacteria like Streptococcus pneumoniae are naturally competent. Other cells, like E. coli, can be made competent by techniques like electroporation which uses electrical pulses to make the cells competent/growing them in high concentrations of calcium.) 13)What is transfection and transduction? How is generalized transduction different from specialized transduction? - Transfection- The transformation of a prokaryotic cell by DNA or RNA from a virus. Used also to describe the process of genetic transformation in eukaryotic cells; Transfection is similar to transformation, except the DNA comes from a phage rather than another bacterium. - Transduction- The transfer of host genes from one cell to another by a virus; Transfer of DNA from one cell to another by a bacteriophage. Generalized transduction: DNA from any portion of the host genome is packaged inside the virion; is a way of transferring bacterial DNA from one bacteria to another via the lytic phase of a bacteriophage. When a transducing phage infects the new cell, the genes are transferred, but the new cell doesn’t get any phage DNA and thus does not lyse. Specialized transduction: DNA from a specific region of the host chromosome is integrated directly into the virus genome; virus can be temperate or virulent; occurs in temperate phages and transfer some of the bacterial genes with it when the prophage leaves the host chromosome. These genes, along with the viral DNA, are then transferred to a new bacterial host. This bacteria is infected with the temperate phage and receives the new bacteria genes in the process. Whatever these genes code for, i.e. antibiotic resistance, are expressed in the new organism. 14)Explain how conjugation works. What are F+ and F- cells? Pili? - Bacterial conjugation (mating): mechanism of genetic transfer that involves cell-to- cell contact; occurs when a cell has the F plasmid (F+) and is able to make a pilus; plasmid-encoded mechanism: Donor cell: contains conjugative plasmid (F+, have fertility plasmid) Recipient cell: does not contain plasmid (F-, don’t have fertility plasmid) - Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together to form a mating pair by retraction of the pilus, which is achieved by de-polymerization. Certain small phages (F-specific bacteriophages) use the sex pilus as receptor and can be seen here attached to the pilus. Has rolling circle replication for transfer of the F plasmid. After conjugation, both have F plasmid (F+) and are able to conjugate, now. 15)What are transposons and insertion sequences? What do they do to the cell they are in? - Transposon- A type of transposable element that carries other genes in addition to those involved in transposition; often these genes confer selectable phenotypes such as antibiotic resistance. Transposase moves any DNA between inverted repeats Insertion of a transposable element generates a duplicate target sequence May include antibiotic resistance (Ex: Tn5 and Tn10) - Insertion sequence- The simplest type of transposable element, which carries only genes involved in transposition. ~1,000 nucleotides long Inverted repeats are 10–50 base pairs. Only gene is for the transposase. Found in plasmids and chromosomes of Bacteria and Archaea. Found in some bacteriophages. - Discrete segments of DNA that move as a unit from one location to another within other DNA molecules are transposable elements. Transposons and insertion sequences are able to move around on the chromosome. When they move in and out of their places on the chromosome they create mutation. They were first observed by Barbara McClintock in her work with maze. Both have inverted repeats at their ends. Learning Objectives Explain how mutations occur in bacteria and what the possible results can be. Diagram the process of replica plating as well as how it is interpreted. Explain how to do and interpret an Ames test. Explain the effects of nucleoside analogs, ethidium bromide, UV light, and gamma rays. Diagram Griffith’s transformation experiment as well as how to interpret it. Compare and contrast transformation, general transduction, specialized transduction, conjugation and Hfr conjugation. MM Qs & As: Figure 10.2: screening for nutritional auxotrophs. Part A: Thymine dimers result from - nonionizing radiation Part B: Which of the following might result in a frameshift mutation? - Benzopyrene Part C: Which of the following describes how 5-bromouracil might create a mutation? - It can replace the base thymine, and can base pair with guanine rather than adenine. Part D: Mutagens - increase the likelihood of mutations in DNA. Part E: A nucleotide-altering chemical - can alter nitrogenous bases of DNA, resulting in incorrect base pairing. A mutant that has a nutritional requirement for growth is an example of a(n) - Auxotroph Figure 10.3: possible effects of base-pair substitution in a gene encoding a protein. Consider a mutation in which the change is from UAC to UAU. Both codons specify the amino acid tyrosine. Which type of point mutation is this? - Silent mutation A "point mutation" refers to mutations involving - a substitution, deletion, or addition of one base-pair. Microinsertions and microdeletions often result in ________ mutations. - Frameshift The mutagens 2-aminopurine and 5-bromouracil are examples of - Nucleotide base analogs The killing of cells by UV irradiation involves - formation of pyrimidine dimers. Ionizing radiation does NOT include - UV rays Intercalating agents, like acridine orange and ethidium bromide, lead to mutagenesis by pushing DNA base pairs apart, which can lead to insertions or deletions. - True Part A: How do restriction enzymes cut DNA sequences? - They cut DNA at sites, called recognition sites, that have specific nucleotide sequences. Part B: In general, how might recombinant DNA technology be used to prevent a genetic disorder caused by a mutation in a single gene? - To insert a desirable gene, remove an undesirable gene, or replace a defective gene with a functioning gene Part C: Which of the following attaches the target gene to a desired location? - DNA ligase Part D: Why would a recombinant DNA molecule be inserted into a host cell? - It can be copied, transcribed, and translated into a desired protein. Part A: Competent cells are cells that - can take up DNA from their surrounding environment and integrate it into their own chromosomes by recombination. Part B: Mice that are injected with only the R strain of Streptococcus pneumoniae - stay healthy, because their immune systems can kill this strain easily. Part C: What characteristic of the S strain allows it to evade the immune system of the mice? - The cells have a capsule Part D: What most likely explains the recovery of live S strain cells from a mouse injected with heat-killed S strain mixed with live R strain cells? - The R strain picked up the S strain DNA, enabling it to produce a capsule. Part E: Which finding is most surprising from Griffith’s experiments? - S strain cells are isolated from the blood of mice infected with heat-killed S strains and live R strains. Figure 10.14: generalized transduction. Figure 10.15: specialized transduction. Part A: What is unique about transduction compared to normal bacteriophage infection? - Transduction transfers DNA from the chromosome of one cell to another. Part B: How is generalized transduction different from specialized transduction? - Generalized transduction is initiated during lytic cycle of a virulent bacteriophage; specialized transduction is initiated during the lysogenic cycle of a temperate bacteriophage. Part C: A transducing phage - contains fragments of the host chromosome instead of the viral genome. Part D: When a transducing phage interacts with a new host cell - the DNA from the previous host can recombine with the new host chromosome. Part A: How does specialized transduction differ from regular lysogeny? - The prophage in specialized transduction carries with it pieces of the host chromosomal DNA. Part B: What happens to the packaged DNA of a specialized transduced phage when it infects a new recipient cell? - The host DNA integrates, with the prophage, into the new recipient chromosome. Part C: How can specialized transduction contribute to the transfer of antibiotic resistance genes in a bacterial population? - The prophage takes an antibiotic resistance gene with it and is packaged with the newly synthesized viral DNA. Part A: What is the function of the conjugation pilus? - It pulls the F+ and F- cells together. Part B: What is required by an F- cell to become an F+ cell? - F plasmid Part C: What is the key difference between donor cells and recipient cells? - An F plasmid Part D: What cellular macromolecule is the fertility factor comprised of? - Nucleic acid Part E: At which point does a recipient cell become an F+ cell? - (5) Formation of the complementary strand of the F factor Part A: How does an F+ cell differ from an Hfr cell? - Hfr strains have the F plasmid integrated into the chromosome. Part B: Why does conjugation between an Hfr strain and an F- strain not result in two Hfr strains? - Conjugation is typically disrupted before the fertility factor can be transferred. Part C: Which of the following is a characteristic of an F+ cell? - Ability to synthesize sex pili, presence of a fertility factor, and ability to mate with an F- cell. Part D: What benefit does the F- strain receive from mating with an Hfr strain? - It acquires new, potentially beneficial genes from the Hfr strain. Which of the following methods may introduce foreign DNA into a recipient? - transformation, transduction, and conjugation The uptake of DNA released from a cell is ________, while transfer of DNA with cell- to-cell contact would most likely result in ________. - transformation / conjugation F+ strains of Escherichia coli - have the F factor as a plasmid. F- strains of Escherichia coli - do not have an F factor. Almost all plasmids are double-stranded DNA. - True Most plasmids are circular rather than linear. - True A plasmid may - replicate independently of the chromosome, integrate into the chromosome, or be transferred cell-to-cell during conjugation. The F (fertility) plasmid contains a set of genes that encode for the ________ proteins that are essential in conjugative transfer of DNA. - Pili Study Guide for Chapter 11 – “ Genetic Engineering and Biotechnology” Vocabulary Beta- galactosidase- breaks down lactose into galactose and glucose & is used in blue/white screening. Blue-white selection- One of the screening techniques that is used to isolate mutants. After the transformation experiment, the cells are plated onto X-gal media that contains ampicillin. Only the cells that contain a plasmid will grow. When the gene-of-interest is inserted into the lac Z gene, it inactivates the gene and the cells are not able to make beta galactosidase. Cells that are able to make beta galactosidase are blue on X-gal media. Cells that are not able to make beta galactosidase, the mutants, are white on X-gal media. Cloning vector- Genetic elements into which genes can be recombined and replicated. DNA fingerprinting- Gel electrophoresis- A tech nique for separation of nucleic acid molecules by passing an electric current through a gel made of agarose or polyacrylamide Genetic engineering- The use of in vitro techniques in the isolation, manipulation, alteration, and expression of DNA or RNA and in the development of genetically modified organisms. Hybridization- Base pairing of single strands of DNA or RNA from two different (but related) sources to give a hybrid double helix. Lac Z gene- codes for beta galactosidase with a polylinker, which is a site on the gene that has multiple restriction/cloning sites which can be cut with restriction enzymes). Molecular cloning- The isolation and incorporation of a fragment of DNA into a vector where it can be replicated. Northern blot- A hybridization procedure where RNA is the target in the gel and DNA or RNA is the probe. Compare with Southern blot and immunoblot. Nucleic acid probe- A strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from a mixture of other nucleic acids. In clinical microbiology or microbial ecology, a short oligonucleotide of unique sequence used as a hybridization probe for identifying specific genes. Polylinker site- a site on the gene that has multiple restriction/cloning sites which can be cut with restriction enzymes). Primer- A short length of DNA or RNA used to initiate synthesis of a new DNA strand. PCR- Artificial amplification of a DNA sequence by repeated cycles of strand separation and replication. rBST- a product primarily given to dairy cattle by injection to increase milk production. Restriction map- A map showing the location of restriction enzyme cut sites on a segment of DNA. Restriction enzyme (sticky or blunt ends)- Enzymes that recognize and cleave specific DNA sequences, generating either blunt or single-stranded (sticky) ends. Restriction site- Sequencing- In reference to nucleic acids, deducing the order of nucleotides in a DNA or RNA molecule. Site-directed mutagenesis- Construction in vitro of a gene with a specific mutation. Somatropin- a form of human growth hormone important for the growth of bones and muscles. Southern blot- A hybridization procedure where DNA is the target in the gel and RNA or DNA is the probe. Compare with Northern blot and immunoblot. Thermocycler- Transgenic animals/organism- A plant or animal with foreign DNA inserted into its genome. Vector- (1) A self-replicating DNA molecule that carries DNA segments between organisms and can be used as a cloning vector to carry cloned genes or other DNA segments for genetic engineering. (2) A living agent, usually an insect or other animal, able to carry pathogens from one host to another. X-gal media- a substitute for lactose used in blue/white screening. Study Questions 1) Describe the basic techniques used in genetic engineering. - Genetic engineering- The use of in vitro techniques in the isolation, manipulation, alteration, and expression of DNA or RNA and in the development of genetically modified organisms; using in vitro techniques to alter genetic material in the laboratory - In vitro means in a test tube and the ultimate goal of genetic engineering is to develop microorganisms that can mass produce products that are coded for by genes. (Ex: genetically engineered insulin.) - Basic techniques include: Restriction enzymes Gel electrophoresis Nucleic acid hybridization Nucleic acid probes Molecular cloning Cloning vectors (Exs: organisms used in GE are E. coli. Bacillus subtilis and Saccharomyces cerevisiae.) 2) What is a restriction enzyme? Give two examples - Restriction enzyme (sticky or blunt ends)- Enzymes that recognize and cleave specific DNA sequences, generating either blunt or single-stranded (sticky) ends; cut the DNA at a restriction site. The restriction enzymes are important in degrading foreign DNA, like viral DNA, that may get into the cell & are used to cut the plasmids and genes-of-interest that are used in genetic engineering; is widespread among prokaryotes and rare in eukaryotes. Recognize inverted repeat sequences (palindromes): Typically 4–8 base pairs long (Ex: EcoRI recognizes a 6-base-pair sequence.) Protect the cell from invasion of a foreign DNA: Destroy foreign DNA. Must protect their own DNA from inadvertent destruction. - (Ex: EcoRI = the first restriction enzyme that was isolated from E. coli & HindIII = isolated from Haemophilus influenza.) 3) How do restriction enzymes know where to cut the DNA? - Recognize inverted repeat sequences (palindromes): Typically 4–8 base pairs long (Ex: EcoRI recognizes a 6-base-pair sequence.) 4) How is a sticky end different from a blunt end? - When the restriction enzyme cuts the DNA, the result is either a “sticky” end or a blunt end. - Sticky end: DNA-ends end with one or more unpaired nucleotides. Blunt end: DNA-ends end with the same complementation base. 5) Why don’t restriction enzymes cut the bacterial DNA? - Bacterial methylases protect the host DNA by methylation (add methyl groups to DNA) in the sequences the restriction enzymes recognize, and the restriction enzymes fail to cleave methylated DNA. The foreign DNA (viral) on the other hand, gets chopped up so the infection is avoided. Source: https://www.quora.com/Why-do-restriction-enzymes-of-bacteria-not-cut-their-own-DNA 6) Why do scientists use gel electrophoresis? Ethidium bromide? - Gel electrophoresis: is a technique that is used to separate proteins and or nucleic acid based on size and charge. You put your samples in little wells within a polyacrylamide or agarose gel. The gel is connected to a power supply. The molecules in the sample move thru the gel and get stuck at various points in the gel based on their size. Smaller pieces move farther. The negative molecules move toward the positive pole. After you have let the gel run for several hours you disconnect it from the power supply. To be able to see the bands you have to stain the gel. - Ethidium bromide: is used to stain DNA gels. Remember that ethidium bromide is a carcinogen so when you work with it you must wear gloves. Once the gel is stained it is placed on a UV light called a transilluminator and the bands will fluoresce. At that point you can take a picture of it and measure the distances between the bands. 7) What is a restriction map? Selection marker? - Restriction map- A map showing the location of restriction enzyme cut sites on a segment of DNA; restriction maps are used to know where the restriction enzymes cut the DNA. - Selectable markers: contain restriction sites. They are easily transformed in the
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