MCB3020 - Exam 3 Study Guide

What is polymerase chain reaction (PCR)?

a technique that rapidly synthesizes billions of copies of a specific DNA fragment from a complex mixture of DNA molecules

• important for diagnostic tests

• used in forensic lab

What are the two types of polymerase chain reaction?

1. end-point PCR - used for making large quantities of desired DNA segment; useful when detecting a pathogen

2. real-time PCR - can quantify the amount of DNA in a sample

What is cDNA synthesis via reverse transcriptase?

• synthesizing complementary DNA (cDNA) from an RNA template

• working with human genes

• no need for RNA processing

What is gel electrophoresis?

method used to separate molecules based on their charge and size

• DNA is acidic so it migrates from the negative to the positive end of the gel

• smaller fragments move faster through the gel

What are vectors?

DNA molecules used to carry foreign DNA into host cells

• ex: plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes

What are the three important features of vectors?

• origin of replication

• region of DNA that contains unique restriction sites

• selectable marker - gene on the plasmid that helps it survive under certain conditions

What are restriction enzymes?

enzymes that recognize and cut specific sequences of DNA

• create blunt ends or sticky ends that are fused with vector and inserted in host cells

What is southern blotting?

• DNA probes detect specific DNA in fragments separated by gel electrophoresis

What is recombinant DNA?

a DNA molecule with segments from different organisms

• allows transfer of genes from eukaryotes to prokaryotes and vice versa

What is the purpose of constructing a genomic library?

• to create a collection of DNA fragments from the entire genome of an organism

What is metagenomics?

• the study of genetic material directly from environmental samples

• helps identify unculturable microbes that cannot grow in laboratory conditions

What is autonomous (self) replication?

• ability of a DNA molecule to replicate independently of the chromosomal DNA within a host cell

• origin of replication

What is DNA cloning?

• use of enzymes and bacteria cells to modify and amplify DNA clone

What is the significance of Ti plasmids?

• integrates into the plant genome and causes a tumorlike growth

• can be used to introduce recombinant DNA into a plant

• ex: Bt toxin - kills pests

What are plasmids?

• small, circular, dsDNA molecules that exist independently of the chromosomal DNA

• used as vectors

What are bacteriophages (phages)?

• viruses that infect and replicate within bacteria

• capsid (protein coat) surrounding DNA or RNA

• tail that injects genes into cells

What are cosmids?

• engineered vectors that are a mix of plasmids and phages

• can carry large DNA fragments

• replicates like a plasmid, but can be packaged into phage capsid and transferred to host cell by transduction

What are artificial chromosomes?

• engineered DNA molecules that are used to clone very large DNA fragments

• ex: yeast artificial chromosome (YAC)

What are the types of genes carried on plasmids?

• antibiotic resistance genes

• virulence genes

• metabolic genes

• bacteriocin genes (kill competing bacteria)

What are the 3 mechanisms of horizontal gene transfer (HGT)?

1. conjugation - direct transfer of plasmids from donor to acceptor through a pilus; most common

2. transformation - uptake of free plasmid DNA from the environment

3. transduction - transfer of plasmid between bacteria via bacteriophages

What is protoplast fusion?

• a technique where the cell walls from two bacteria cells are removed to produce protoplasts, which are then fused together

What is electroporation?

• a method that uses an electric current to create temporary pores in the cell membrane to introduce recombinant DNA into host cells

What are the therapeutic applications made possible by biotechnology?

• human enzymes and proteins such as insulin

• subunit vaccines - made from pathogen proteins in genetically modified yeasts

• gene therapy (ex: gene editing)

• vaccines

What is gene silencing?

• process by which the expression of a specific gene is reduced or turned off

• small interfering RNAs (siRNAs) bind to mRNA and destroy it

• could provide treatments for a wide range of diseases

What role does biotechnology play in herbicide resistance?

• introducing herbicide resistance genes into crops

• increases crop yield

What is genome shotgun sequencing?

• method used to determine the complete DNA sequence

• break the genome into many small fragments

• sequence the fragments

• use computer to assemble the sequences

What is Cas9 genome editing (CRISPR)?

• tools for altering genomes

• allows direct modification of genomic DNA in any cell

• Cas9 - endonuclease that cuts both strands of target DNA

• CRISPR - a natural bacterial immune system

What are ribonucleoproteins?

• complexes made of RNA molecules bound to proteins

• Cas9 protein and RNA form a ribonucleoprotein that recognizes and cuts specific DNA sequences

What is genomics?

the study of molecular organization or genomes, their information, and gene products they encode

What are the 3 areas of genomics?

1. structural genomics - studies the physical nature of genomes

2. functional genomics - studies the functions of genomes

3. comparative genomics - compares genomes of different organisms

What is bioinformatics?

• uses computer science, math, and stats to generate data on genome content, structure, and arrangement

• also data on protein structure and function

• uses annotation to determine location of genes

What is In Silico Analysis in bioinformatics?

using computer simulations and methods to analyze genome and protein sequences

What is genome annotation in bioinformatics?

• process of identifying and locating genes in the genome map

• identifies each open reading frame in genome

• uses databases to assign tentative function of gene

Compare paralogs to orthologs?

• Paralogs - genes found alike within the same species that likely arose from gene duplication

• Orthologs - genes very similar in different species that are predicted to have the same function

What are motifs?

• short patterns of amino acids that may represent a functional unit within a protein, such as the active site of an enzyme

• phylogenetically well conserved

What is the Microarray System?

• powerful laboratory tool used to study gene expression or detect specific DNA or RNA sequences

• samples used: tissue, DNA, blood, saliva, etc.

What is proteomics?

• study of the entire set of proteins produced by an organism (proteome)

• provides info about genome function not available from mRNA studies

What role does comparative genomics play in the analysis of pathogenic microbes?

• provides information about virulence and evolution

• provides information about potential targets for therapy or vaccines

What is 2D gel electrophoresis?

technique used to separate proteins in two steps

• separation by charge: proteins are placed in a pH gradient, and they migrate until net charge is zero (isoelectronic point)

• separation by size: smaller proteins move faster; proteins are coated with negative charge

What is bioprospecting?

process of searching for useful biological resources in nature

• ex: plants, animals, microbes, or genetic material

What is high-throughput screening (HTS)?

rapid and automated technique used to test millions of samples for a specific biological activity

How are viruses different from living things?

• non-cellular

• DNA or RNA (never both)

• reproduce inside host cells

What are methanogens?

• group of archaea that produce methane

• live in anaerobic environments rich in organic matter

• positive impacts: nutrient cycling, digestion

• negative impact: methane is a greenhouse gas

What are the impacts of chemolithotrophs on the environment?

• fix CO2 into organic molecules

• crucial in nutrient cycling

• soil fertility

What is the difference between methanogens and methanotrophs?

• methanogens

  • archaea

  • strict anaerobes

  • produce methane

• methanotrophs

  • bacteria

  • mostly aerobes

  • consume methane

What are 3 anaerobic environments (organic-rich) where methanogens are found?

• rumen (stomachs) of animals

• anaerobic sewage sludge

• within anaerobic protozoa

What are halophilic archaea?

• require high salt concentrations (> 1.5 M salt)

• some are extreme acidophiles or live in extreme alkali conditions

What are thermophilic archaea?

• grow best at high temperatures (70-110 Celsius)

• some can metabolize sulfur for energy

• found in marine hydrothermal vents (aka black smokers)

What role do microbes play in the carbon cycle?

• cyanobacteria and algae fix atmospheric CO2 into organic carbon (sugars)

• bacteria and fungi break down dead organisms and organic waste which releases CO2 back into the atmosphere through microbial respiration

• methanogens converts organic compounds into methane

What role do microbes play in the nitrogen cycle?

• nitrogen fixation - nitrogen fixing bacteria convert N2 into ammonia or ammonium

• ammonification - decomposer bacteria and fungi break down waste and minerals into ammonia

• nitrification - nitrifying bacteria turns ammonia into nitrite and then nitrate

• denitrification - denitrifying bacteria converts nitrate back into N2

What is the overall purpose of the sewage treatment process?

to reduce biological oxygen demand (BOD) before release into the environment, since sewage is high in organic matter

What are the 3 stages of the sewage treatment process?

1. Primary treatment - remove large solids through screening and sedimentation; microbes not involved

2. Secondary treatment - microbes break down dissolved organic matter; removes the most BOD

3. Tertiary treatment - using filtration and disinfection to remove remaining nutrients and pathogens

What is the role of the anaerobic sludge digester in sewage treatment?

• microbes break down organic matter in a sealed, oxygen-free tank

• produces methane which is used as fuel (electricity and heat) to power the plant

What is the septic tank treatment?

• simple, primary treatment of sewage from areas with low population density

• effluent (partially clarified wastewater) from the holding tank is piped into a drainage field

• anaerobic bacteria breaks down the organic matter in the sludge

What is composting?

aerobic microbial breakdown of organic matter into compost that can be used as fertilizer

What is bioremediation?

process that uses microbes to remove pollutants from the environment

• microbes metabolize pollutants and convert them into harmless products like water and CO2

• ex: oil spill cleanup

What are the three basic principles of antimicrobial therapy?

• selective toxicity - kill pathogens without damaging the host

• reach the site of infection in sufficient, inhibitory concentrations

• penetrate and bind to target while avoiding inactivation by microbial enzymes and extrusion by efflux pumps

What are the sources of antibiotics?

• Naturally produced by some soil bacteria and fungi to eliminate the competition in soil where nutrients are scarce

• Bacteria such as Bacillus and Actinomycetes

• Fungi such as Penicillium

Why do some microorganisms produce antibiotics in nature?

• defense mechanism

• compete with competitors

What are chemotherapeutic agents?

• antimicrobial drugs used to treat diseases by inhibiting the growth of disease-causing organisms without harming the host

What are the 4 categories of antimicrobial drugs based on their mechanisms of action?

1. inhibitors of cell wall synthesis

   • ex: penicillin and cephalosporin

2. inhibitors of protein synthesis

   • ex: tetracyclines, erythromycin, streptomycin, chloramphenicol

3. inhibitors of nucleic acid synthesis

   • ex: rifamycin and ciprofloxacin

4. inhibitors of cell membrane function

What are antimetabolites?

antimicrobial agents that inhibit the synthesis of essential metabolites by mimicking natural substrates and competing for enzyme binding sites

Compare sulfanilamide drugs and trimethoprim.

• Sulfa drugs - structural analog of PABA that competes with PABA and prevents the synthesis of folic acid

• Trimethoprim - inhibits dihydrofolate reductase, prevents activation of folic acid, treat UTI, combined with sulfa drugs to increase efficacy

What antibiotics affect DNA replication and how do they work?

• Ciprofloxacin (a Fluoroquinolone)

• binds to topoisomerase, leading to the accumulation of DNA breaks

What antibiotics affect RNA transcription and how do they work?

• Rifamycin

• binds to RNA polymerase and blocks the initiation of transcription

• used to treat TB

What is a beta-lactam ring?

• a four-membered cyclic amide structure that is essential for inhibitors of cell wall synthesis

• binds to enzymes which are involved in forming the peptidoglycan layer

What do penicillin, cephalosporin, bacitracin, and vancomycin have in common?

• all four inhibit bacterial cell wall synthesis

• especially effective against Gram-positive bacteria

What is the role of beta-lactamase in bacterial resistance to antibiotics?

enzyme produced by penicillin-resistant bacteria that breaks open the beta-lactam ring, making it ineffective

Why is protein synthesis an excellent target against some infectious diseases, but not others?

• antibiotics specifically target bacterial ribosomes which have 70S ribosomes (50S +30S), while human cells have 80S ribosomes

• however, viruses do not have their own ribosomes and rely on their host’s

• fungi are eukaryotic and have 80S ribosomes

What is therapeutic dose?

the minimum amount of drug needed for clinical treatment without harming the host

What is the therapeutic index?

• the ratio of toxic dose to therapeutic dose

• the higher the index, the safer the drug

What are the mechanisms of bacterial resistance to antimicrobial drugs?

• prevent entrance of drug by reducing permeability

• efflux pumps - proteins that pump out antibiotics

• drug inactivation

• modification of target enzyme or organelle to reduce drug binding

• use of an alternative pathway or increased production of target metabolite

What are R plasmids (resistance plasmids)?

• carry multiple antibiotic resistance genes

• plasmid can be transferred to other cells by conjugation, transduction and transformation

What are composite transposons?

• contain multiple antibiotic resistance genes

• can move rapidly between plasmids and through a bacterial population

What are the common reasons for increasing drug resistance and emergence of superbugs?

• using outdated antibiotics

• using antibiotics for the common cold or other inappropriate conditions

• using antibiotics in animal feed

• failing to complete the prescribed regimen

• using someone else’s leftover prescription

What are anti-fungal drugs?

• Polyenes - bind to ergosterol, disrupting cell membrane function

  • Nystatin - most used antifungal drug

  • Amphotericin B - produced by Streptomyces and toxic to the kidneys

• Azoles - block ergosterol synthesis and inhibits cell membrane synthesis

• Griseofulvin - produced by penicillium fungi and inhibits microtubule formation

What are antiviral drugs?

• Acyclovir - a nucleoside analog that resembles Guanine and inhibits herpes replication

• Tamiflu - anti-influenza agent that treats influenza A and B

  • ex: Amantadine - effective against influenza A

What are antiprotozoal drugs?

• Chloroquine – treats malaria by preventing the parasite from detoxifying heme metabolite

• Metronidazole – treats anaerobic infections such as trichomonas

What antibiotics inhibit protein synthesis?

• chloramphenicol - bind to 50S ribosomal subunit, blocks peptide bond synthesis

• Macrolides (e.g., Erythromycin) - bind to 50S subunit, inhibits translocation of the ribosome along the mRNA

• Aminoglycosides (e.g., Streptomycin) - bind to 30S subunit, cause misreading of mRNA, lead to nonfunctional proteins

• Tetracyclines (e.g., doxycycline) - bind to 30S subunit, prevents addition of new amino acids, treat severe acne

What is the Kirby-Bauer test?

• tests the effectiveness of antibiotics

• paper disks with antibiotics are placed on an agar

• zones of inhibition determines the sensitivity and resistance of the organisms to the antibiotic

What is the Etest?

• Etest strips contain a gradient of an antibiotic

• intersection of elliptical zone of inhibition with the strip indicates the MIC

What is the serial dilution assay?

method used to gradually dilute a solution to estimate the number of viable bacterial or viruses in a sample to test effectiveness

• determines the minimum inhibitory concentration (MIC) - the lowest concentration of drug that inhibits the growth of the pathogen

How do synergistic drug interactions work?

• two drugs work together to produce a greater effect than the the effect of either alone

• ex: sulfa drugs and trimethoprim (both antimetabolites)

How do antagonistic drug interactions work?

• two drugs working together create a lesser effect than the effect of either alone

• ex: tetracycline sometimes interferes with the activity of penicillin

Why are immunosuppressed individuals given antifungal agents?

they have weakened immune systems, making them highly vulnerable to fungal infections

Why are there far fewer antiviral drugs than antibacterial drugs?

• difficult to target the virus without harming the host

• viruses have few virus-specific enzymes

• viruses mutate quickly

What are reverse transcriptase (RT) inhibitors?

• block the activity of RT which HIV uses to copy its RNA into DNA

• nucleoside RT inhibitors - mimic nucleotides, preventing RT from synthesizing DNA

• non-nucleoside RT inhibitors - bind directly to change RT’s shape

What are protease inhibitors?

mimic the peptide bond that is normally attached by the HIV protease so that the virus produces non-infectious virus particles

What are fusion inhibitors?

prevent HIV entry into human immune cells

What are key characteristics of viruses?

• sub-microscopic: must be observed with electron microscopes

• obligate intracellular parasite: cannot replicate on their own and do not carry the enzymes or ribosomes needed

• host specificity: specific to certain cell types

What are the different components of a virus?

1. nucleic acid

   • DNA or RNA, single or double stranded

   • all viruses must have this

2. capsid

   • protein coat that protects nucleic acid

   • helps the virus attach to host cells

   • all viruses must have this

3. envelope

   • outer, flexible membrane composed to lipid and proteins

4. spike

   • protein projections on surfaces of viruses, helps with recognition and attachment to host

What are the steps of virus replication?

1. attachment (adsorption) - bind to specific receptors on host

2. entry - fuse with membrane or endocytosis

3. uncoating of genome - release nucleic acid inside host as capsid breaks down

   • due to low pH

4. synthesis - making virus components

5. assembly (maturation) - components assemble into new virus

6. release - virus leaves the cell as virions

What are cytopathic effects (CPE)?

visible changes that occur in host cells due to viruses

• Plaque (clear zone) formation in phage

• Nuclear or cytoplasmic enlargement

• Changes in membrane, fibroblast and viral budding

• Syncytia formation - multiple infected cells fuse together

• Inclusion bodies

• Vacuole formation or lysosomal leakage

What are the two types of capsid shapes?

• Helical capsids (spiral-shaped) - rod like

• Icosahedral capsids (geometric) - made up of 20 triangular faces and 12 vertices

What are ways viruses enter the body?

• Respiratory tract - through inhalation

• Alimentary tract - through contaminated food, water, or hands

• Skin

• Genital tract - through sexual contact

• Conjunctiva (eye)

• Parenteral route - unconventional, but direct entry into tissues and bloodstream, bypassing skin

What are ways viruses exit from the body?

• Respiratory and oropharyngeal secretions - coughing, sneezing, talking, etc.

• Feces and urine

• Skin - shed from lesions, sores, or blisters

• Breast milk

• Genital secretions

• Blood

• No shedding

What are ways viruses spread in the body?

• Local spread of epithelial surfaces

• Subepithelial invasion/lymphatic spread - penetrate into underlying tissues

• Viremia - spread by the bloodstream

• Invasion of skin, CNS, other organs

• Invasion of the fetus

What are permissive or productive infections?

host cell allows full viral replication

What are latent infections?

virus becomes dormant for a period, then can reactivate later

What are chronic infections?

virus continues to replicate and cause damage over time; less viruses produced