BIO2200 Final Exam – Flashcards

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Microbiology
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The study of microorganisms.
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Microorganisms
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All single-celled microscopic organisms and include all viruses, which are microscopic but not cellular.

They are independent entities that carry out their life processes independently of other cells.
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Properties of cellular life
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1. Metabolism: The take up of nutrients from the environment and transformation of them into new cell materials and waste products.

2. Growth: The increase in cell number due to cell division.

3. Evolution: the precess of descent with modification in which genetic variants are selected based on their reproductive fitness.

4. Motility: Allows cells to move away from danger or unfavorable conditions and to exploit new resources or opportunities.

5. Differentiation: changing based on chemical signals in their environment.

6. Communication: responding to chemical signals in their environment.
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Cell
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The fundamental unit of life.

A single cell is an entity isolated from other such entities by a membrane.
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Habitat
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The immediate environment in which a microbial population lives.
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Microbial communities
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Where populations of cells interact.

The diversity and abundance of these communities is controlled buy the resources (foods) and conditions (Temperature, pH, oxygen content, and so on...)
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Ecosystem
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All living organisms, together with the physical and chemical components of their environment.

Ex: aquatics, terrestrial, and other organisms such as plants and animals.
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LUCA
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Last Universal Common Ancestor...
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Cells first appeared on earth...?
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3.8-3.9 billion years ago...

The earth is 4.6 billion years old.
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The first cells
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Methanogens: produce only methane

Phototrophs: harvest energy from sunlight

Bacteria
Archaea
Eukarya (the ancestors of the plants and animals)
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Pathogens
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Microorganisms which can cause infectious disease.

Influenza
Pneumonia
Gastroenteritis
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Genomics
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The science of identification and analysis of genomes which has greatly enhanced biotechnology.
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Robert Hooke
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English
1664
Discovered microorganisms (fungi)
Famous book, Micrographia
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Antoni van Leeuwenhoek
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Dutch
1684
Discovery of bacteria
"wee animacules"
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Edward Jenner
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English
1798
Vaccination (smallpox)
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Louis Pasteur
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French
1800s
Mechanism of fermentation
Defeat of spontaneous generation
Rabies and other vaccines
Principles of immunizations
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Ferdinand Cohn
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German
1876
Discovery of endospores
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Robert Koch
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German
Late 1800s
Koch's Postulates
Pure culture microbiology
Discovery of agents of TB and cholera
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Spontaneous Generation
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From biblical times, says things arise spontaneously from non-living things.

Pasteur debunked this theory by protecting food in a sterile environment to keep it from being contaminated by organisms in the air and putrefying it. (swan-necked flask experiment)
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Koch's postulates
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For definitively linking a specific microorganism to a specific disease:

1. The disease-causing organism must always be present in animals suffering the disease but no in healthy animals.

2. The organism must be cultivated in a pure culture away from the animal body.

3. The isolated organism must cause the disease when inoculated into healthy susceptible animals.

4. The organism must be isolated from the newly infected animals and cultured again in the laboratory, after which it should be seen to be the same as the original organism.
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Chemolithotrophy
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The oxidation of inorganic compounds to yield energy.

The organisms are called chemolithotrophs, and they obtain their carbon from CO2, thus, they are autotrophs.
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Resolution
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The ability to distinguish two adjacent objects as distinct and separate.
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Differential stains
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Stains that render cells a different color, like the Gram stain, which can differentiate bacteria into being either Gram positive or Gram negative.
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Gram positive
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Purple-violet

Cell wall is 90% peptidoglycan, laid down in "cables".

Has acidic component called teichoic acids embedded in their cell walls and are the reason for the overall negative charge of the cell surface.
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Gram negative
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Pink

Cell wall only 10% peptidoglycan, rest is "outer membrane", which is effectively a second lipid bilayer, but also contains polysaccharides as a complex. Also known as the lipopolysaccharide layer (LPS).

Composition from bottom to top:
Cytoplasmic membrane
Periplasm
Peptidoglycan
Outer phospholipid bilayer
Lipopolysaccharides
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The process of staining cells for microscopic observation
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Spread culture in thin film over slide
Let dry in air
Heat fix with flame
Flood with stain
Rinse and Dry
Place drop of oil on slide, examine with 100x objective lens.
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Cytoplasmic membrane
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A permeability barrier that separates the inside of the cell from the outside, used for transport, and conservation of energy.

Composed of phospholipid by layer with membrane proteins.
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Cytoplasm
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The inside of the cell.

An aqueous mixture of macromolecules (proteins, lipids, nucleic acids, and polysaccharides), small organic molecules (mainly precursors to macro molecules), various inorganic ions, and ribosomes.
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Ribosomes
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The cell's protein-synthesisizing structures in the cytoplasm.
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Cell wall
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Lends structural strength to a cell.
Relatively permeable and located outside the membrane.
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Viruses
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Not cells. Much smaller and lack many cellular attributes.
Static and stable, unable to change or replace its parts by itself.
Cannot replicate until it infects a cell, since they have no metabolic capabilities of their own.
Only contain a single form of nucleic acid.
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Phylogeny
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The evolutionary relationships between organisms.
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Endosymbiosis
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The theory of how this stable arrangement of cells led to the modern eukaryotic cell with organelles, with mitochondria and chloroplasts developing from Bacteria.
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Chemoorganotrophs
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Organisms that conserve energy from organic chemicals.
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Heterotrophs
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Require organic compounds as their carbon source.
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Autotrophs
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Use carbon dioxide as their carbon source.
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Eukaryote
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A cell having a membrane-enclosed nucleus and usually other membrane-enclosed organelles.
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Nucleoid
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The aggregated mass of DNA that constitutes the chromosome of cells of Bacteria and Archaea
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Proteobacteria
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A large phylum of Bacteria that includes many of the common gram-negative bacteria such as E. coli.
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Protists
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Algae and protozoa.
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Plasmids
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Extrachromosomal genetic element nonessential for growth.
Generally in Prokaryotes.
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Coccus
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(cocci)
Bacterium with spherical or ovoid shape.
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Bacillus
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(rod)
A bacterium with a cylindrical shape.
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Spirilla
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Rods that twist into spiral shapes.
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Surface-to-Volume Ratio
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As a cell increases it's size, its S/V ratio decreases.

A higher S/V ratio:
-Supports a faster rate of nutrient exchange, and therefore a faster growth rate
-Allows for more evolution based on cell mutations during replication, since a smaller cell can divide faster.
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TRANSPORT SYSTEM: Simple transport
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Consists only of a membrane-spanning transport protein.
Energy-driven, by the accumulation of lactose in the cytoplasm against the concentration gradient.

Ex: Lac Permease in E. coli
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TRANSPORT SYSTEM: Group translocation
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The phosphotransfrase system!
A type of transport in which the substance transported is chemically modified during its uptake across the membrane.
Consists of a family of proteins that work together:
Five proteins are necessary to transport any given sugar.
The proteins are alternately phosphorylated and dephosphorylated until the actual transporter, Enzyme IIc, phosphorylates the sugar during the transport event.

Energy for this system comes from the energy-rich compound phosphoenolpyruvate, which is a key intermediate in glycolysis, a major pathway for glucose metabolism.
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TRANSPORT SYSTEM: ABC System
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A transport system that employs periplasmic binding proteins along with a membrane transporter and ATP-hydrolyzing proteins.

A - ATP
B - Binding
C - Cassette
(A structural feature of proteins that bind ATP)

Exists for the uptake of organic compounds such as sugar and amino acids, inorganic nutrients such as sulfate and phosphate, and trace metals.

Periplasmic binding proteins have very high substrate affinities and can bind them even at very low concentrations. Then it interacts with its respective membrane transporter to transport the substrate into the cell via ATP hydrolysis.
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Uniporter
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Protein that transports a molecule unidirectionally across the membrane, either in or out.
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Symporter
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Cotransporter protein that transports one molecule along with another substance, typically a proton.
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Antiporter
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Protein that transports one molecule into the cell while simultaneously transporting a second molecule out of the cell.
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Protein export
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Exported through and inserted into prokaryotic membranes by the activities of other proteins called translocases.

Sec(retory) stystem transports proteins by recognizing them based on the way they are tagged.

Important because many bacterial enzymes are designed to function outside the cell.
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Peptidoglycan
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A polysaccharide composed of two sugar derivative and a few amino acids, and either lysine or the structurally similar amino acid analog, DAP.

Can be destroyed by certain agents, like lysozyme.

Only present in species of bacteria.
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Bright-field microscope
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Employs a microscope with a series of lenses to magnify and resolve the image.

Limitation: the lack of contrast between cells and their surroundings. Can be overcome with staining or alternative forms of light microscopy, like phase contrast or dark field.
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Electron microscope
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Far greater resolving power than light (up to 0.2nm)

Transmission: primarily used for observing internal cell structure
Scanning: used for observing the surface of specimens
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All microbial cells share certain basic structures, such as:
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Cytoplasmic membrane and ribosomes.

Most bacterial cells have a cell wall.
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Genome
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A cell's complement of genes, which governs its properties.
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Comparative rRNA gene sequencing has defined three domains of life:
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Bacteria
Archaea
Eukrya

Molecular sequence comparisons have shown that organelles of Eukrya were originally Bacteria.
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LPS
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Lipopolysaccharide layer, which makes up the majority of a gram negative cell wall.

Some carry toxic outer membrane components (endotoxins).
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Periplasm
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Space located between the outer surface of the cytoplasmic membrane and the inner surface of the outer membrane. Contains several different classes of proteins, like hydrolytic enzymes for the initial degradation of food molecules, binding proteins for the transportation of substrates, and chemoreceptors for the chemotaxis response.
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Porins
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Embedded in the outer membrane of gram negative bacteria, they function as channels for the entrance and exit of solutes.

Nonspecific porins form water-filled channels through which any small substance can pass.

Specific porins contain a binding site for only one or a small group of structurally related substances.

They are transmembrane proteins.
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Archaea cell walls are made up of...
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S-layer, the most common, which is paracrystalline consisting of interlocking protein or glycoprotein molecules that show an ordered appearance under the microscope.

Pseudomurein, a polysaccharide , although some other archaea cell walls are made of different polysaccharides, but no peptidoglycan.
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Capsule
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Another cell surface structure, a layer organized in a tight matrix that excludes small particles, such as India ink. Typically adhere firmly to the cell wall.
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Slime layer
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Another cell surface structure, a layer that is more easily deformed, will not exclude small particles, and is more difficult to see. Loosely attached and can be lost from the cell surface.
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Fimbriae
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Filamentous structures composed of protein that extend from the surface of a cell. Enable cells to stick to surfaces.
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Pili
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Filamentous structures composed of protein that extend from the surface of a cell. Typically longer and only one or a few present. Can be receptors for certain types of viruses.

Facilitate genetic exchange between cells in a process called conjugation.

Adhere pathogens to a specific host tissue and subsequent invasion.

Allow for twitching motility.
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Cell inclusions
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Often present in prokaryotic cells. Function as energy reserves and as reservoirs of structural building blocks.

PHB (poly-B-hydroxybutyric acid) for carbon
Glycogen for carbon
Magnetosomes for iron
Polyphosphate and sulfur
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Endospores
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Highly differentiated cells that are extremely resistant to heat, harsh chemicals, and radiation. They allow a cell to survive unfavorable circumstances or as a dormant stage of the cell's life.

Converts back to vegetative cell in three steps:
1. Activation: heated
2. Germination: placed in presence of certain nutrients
3. Outgrowth: involves swelling due to uptake of water and synthesis of RNA, proteins and DNA. Cell emerges from broken endospore and begins to grow.
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Flagellum (flagella)
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Functions to allow "swimming" motility of cells by rotating to push or pull the cell through a liquid medium.

Long, thin appendages free at one end and attached to the cell at another, even in different places.

Polar flagellation, attached to one or both ends of a cell. When tufts of flagella at one end, its called lophotrichous. When tufts at both ends, called amphitrichous.
When at many locations, called peritrichous.

Also, gliding motility, but not be flagella
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Motile bacteria respond to...
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Physical and chemical gradients in their environment.
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Chemotaxis
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Directed movement of an organism toward or away from a chemical gradient.
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Phototaxis
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Movement of an organism toward light
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Catabolic
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Metabolic reaction where energy is RELEASED
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Anabolic
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Metabolic reaction where energy is REQUIRED
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Essential chemical components of the cell
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C
H
O
N

P
S
K
Mg
Ca
Na
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Micronutrients
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Fe
B
Cr
Co
Cu
Mn
Ni
W
Zn
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Culture media
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Nutrient solutions used to grow microorganisms in the lab.

Defined media: prepared by adding precise amounts of highly purified inorganic or organic chemicals to distilled water so that the exact composition is known.

Complex media: employ digest of microbial, animal, or plant products, such as casein, beef, soy, yeast.
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Ribosomes
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Macromolecular complexes composed of RNA and protein and the site of protein synthesis in all organisms.
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What is required to overcome the limitation in cell size?
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Compartmentalization
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Sterols
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Made only by eucaryotes and incorporated into their membranes to give them strength but makes it less flexible. One group of prokaryotes have sterols in their membranes-the mycoplasmas.
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Hopanoids
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The bacterial equivalent of sterols. They have a
structure similar to sterols and may well be involved in providing strength to the membrane.
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What are the 3 ways molecules move across membranes?
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Osmosis-movement of water
Diffusion-high to low concentration
Transport by membrane proteins
a. Simple transporters
b. Group translocation
c. ABC transporters
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How does penicillin function
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inhibits the formation of peptidoglycans
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Carrier Mediated transport shows saturation kinetics. Define saturation kinetics.
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At some point, all of the transport proteins are busy transporting molecules and adding more to the growth medium will not affect the rate of entry in the cell
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Carrier mediated transport allows accumulation of molecules ...
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AGAINST their gradient
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What does a lysozyme break in bacteria?
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B,1-4 linkage of the peptidoglycan
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Lipid A
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attaches the polysaccharide to the phospholipid bilayer
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Where does the energy use in a flagella come from?
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proton motive force.
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How does cross linkage occur in Gram (+) bacteria?
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It occurs by way of a peptide interbridge
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Selective Media
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contains compounds that selectively inhibit the growth of certain microbes
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Differential Media
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contains an indicator that allows for differentiation of particular chemical reactions during growth
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Free Energy (G)
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the energy released that is available to do useful work.

Exergonic Reactions: release energy (negative deltaG)
Endergonic Reactions: requires energy (postive deltaG)
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What two things does a catalyst do?
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1) lowers the energy of activation of a reaction
2) increases the rate of reaction
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Enzymes increase the rate of reaction by how many times?
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10^8 to 10^20
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Coenzymes
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loosely bound to enzymes; may be associated with more than one type of enzyme (NAD+/NADH)
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Cellulase
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enzyme that break down cellulose
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Glucose Oxidase
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catalyzes the oxidation fo glucose
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Ribonuclease
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decomposes nucleic acid
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What is the primary electron donorWhat is the terminal electron acceptor?
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NAD+/NADH
O2
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What are two mechanisms for energy conservation known in chemoorganotrophs?
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1) Fermentation
2) Respiration
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Pyruvate
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The end product of glycolysis
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Which two roles does the Citric Acid Cycle play?
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1) Bioenergetic:Produces NADH and FADH for use in electron transport chain.

2) Biosynthetic: produces compounds that are used in the synthesis of other important biomolecules.
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Substrate Level phosphortylation
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an inorganic phosphate group gets added to an intermediate in the biochemical pathway and eventually gets transferred from ADP to for ATP
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Oxidative Phosphorylation
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cytoplasmic membrane energized by the proton motive force dissipates energy that allows ADP and PO4 to form ATP
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What three classes of protein electron carriers does aerobic respiration use?
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NADH dehydrogenases (accept 2e- or 2H+)
Flavoproteins (accept 2e- or 2H+)
Cytochromes (accept single e-)
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Quiniones
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another type of e- carrier that are non- protein accept 2e- or 2H+
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Proton Motive Force
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When e- are transported through the e- transport chain, protons are extruded to the outside of the membrane
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ATP Synthase (ATPase)
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large membrane enzyme catalyst complex that is responsible for the conversion of the proton motive force (pmf) into the production of ATP
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ATPase catalyzed ATP synthesis is referred to as what in respiratory systems
In phototropic systems?
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oxidative phosphorylation

Photo-phosphorylation
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What is the primary difference between anaerobic and aerobic respiration?
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Anaerobic respiration uses e- acceptors other than O2, such as NO3-, Fe3+, SO42-, and CO32-
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Photoautotrophy
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Use light as an energy source in the process of photosynthesis
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What are the five steps of Binary fission?
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1) DNA Replication
2) Cell Elongation
3) Septum Formation
4) Formation of Distinct cell walls
5) Cell Separation
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Divisome
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Division apparatus in the cell orchestrates synthesis of new cytoplasmic membrane and cell material in both directions until the cell reaches twice its original size
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How is the divisome formed?
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formed by FtsZ proteins in a ring around the cell center FtsA, FtsI, and ZipA are attracted to the ring structure and associate with FtsZ
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DNA replication occurs prior to what?
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FtsZ ring formation
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FtsK
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helps to pull apart daughter cells following elongation
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What are three characteristics of MreB?
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1) the major shape determining protein in prokaryotes
2) very similar to actin protein in eukaryotic cells
3) not found in coccus-shaped bacteria (default shape is spherical)
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Autolysic enzymes
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in divisome complex create openings in the cell wall so that peptidoglycan can be distributed between daughter cells
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What does autolysin cut to make new peptidoglycan?
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It cuts pre-existing peptidoglycan and inserts peptidoglycan precursors (such as muramic acid and glucosamine)
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Bactoprenol
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lipid carrier molecule that helps in the insertion of glucosamine and muramic acid in new peptidoglycan transports precursors across cell membrane for incorporation into the growing point of the cell wall
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Transpeptidation
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Final step of cell wall synthesis formation of peptide cross links between muramic acid residues in adjacent glycan chains
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Penicillin
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inhibits transpeptidation binds to Fts proteins and render them catalytically inactive
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Generation time
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the time required for cell population to double
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What are the four phases of exponential growth?
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1) Lag Phase: may be brief or extended depending on history of culture and growth factors

2) Exponential Phase: may be brief or extended depending on nutrients smaller cells grow faster than larger cells

3) Stationary Phase: do not increase or decrease in cell number essential nutrients being used up and waste products are accumulating

4) Death Phase: Cells begin to slowly die off
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How is microbial growth measured?
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measured by the change in the number of cells over time

Direct Count:cell counts done microscopically mthat measure the total number of cells (living or dead) in a population

Plate Counts: only measures viable cells. Requires 1) Spread Plate method 2) Pour Plate method
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What is the typical cardinal temperature range for microbes?
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30-40 degrees celsius

Minimum Temperature: below this no growth occurs as cytoplasmic membrane malfunctions
Optimal Temp: growth is most rapid
Maximum Temp: above this growth is not possible as proteins denature
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What are the four temperature classes of microbes?
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1) Psychorophiles (low temp optima (4 deg C)
2) Mesophiles (midrange temp optima (39 deg C)
3) Thermophiles (high temp optima (60 deg C)
4) Hyperthermophiles (very high temp optima (88 deg C)
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How do psychrophiles adapt to their cold environment (four reasons)?
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1) Produce enzymes that function optimally at cold temperatures
2) higher ratio of a-helix to b-sheets in enzymes
3) higher content of polar versus hydrophobic amino acids in enzymes
4) cytoplasmic membranes have a higher content of unsaturated fatty acids, allowing membrane transport to proceed unhindered
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How do thermophiles adapt to their hot environment (four reasons)?
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1) have key amino acid changes in enzymes and proteins that prevent denaturation
2) increased number of ionic bonds between (+) and (-) charged amino acids
3) cytoplasmic membranes have a higher content of saturated fatty acids
4) proteins have highly hydrophobic interiors which prevents unfolding
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What are the three environmental factors that effect microbial growth?
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1) pH
2) Osmolarity
3) Oxygen
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Types of -philes
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Acidophiles: Microbes that grow at a pH of <5
Alkaliphiles: Microbes that grow at a pH of >9
Halotolerant microbes: tolerate environments with a low aw but do not grow well
Extreme Halophiles: grow in extremely salty environments (15-30% NaCl)
Osmophiles: grow in environments with high sugar concentration
Xerophiles: grow in very dry environments
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What are the three groups of aerobes?
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1) Obligative Aerobe
2) Faculative Aerobe
3) Microaerophillic
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What are the two types of anaerobes?
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1) Aerotolerant
2) Obligate
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What are the four toxic forms of oxygen?
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1) Singlet (1O2)
2) Superoxide anion (O2-)
3) Hydrogen peroxide (H2O2)
4) Hydroxyl radical (OH)
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What are the enzymes that destroy hydrogen peroxide?
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Catalase and Peroxidase
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What is the enzyme that destorys superoxide?
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Superoxide dismutase
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Three properties of water
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1) Excellent Solvent
2) Polar compound
3) Outstanding cohesiveness
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Amphiphatic
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Possess both hydrophobic and hydrophillic properities, as in lipids
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Three components of Nucleotide
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1) 5-carbon sugar
2) Nitrogen base (attached to C1 carbon via glycosidic bond)
3) molecule of phosphate (PO4) (attached to C5 carbon)
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Structurally how does DNA differ from RNA?
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DNA has an H attached to the C2 carbon, while RNA has an OH
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Phosphodiester bond
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Phosphodiester bond links C5 carbon phophate group to C3 carbon of another nucleotide
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Peptide bonds
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link together amino acids carboxyl carbon of one amino acid is bound to the amino nitrogen of another amino acid
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Protein Structures
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Primary: linear chain of amino acids covalently bonded to one another

Secondary: involves interaction between the R groups of the amino acids in the polypeptide or atoms in the peptide bonds involves hydrogen bonding to a large degree

Tertiary: involves folding of the molecule and formation of exposed regions

Quaternary: Seen in proteins that consist of two or more polypeptide chains
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The three key processes of macromolecular synthesis
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1) DNA replication
2) transcription: synthesis of RNA from a DNA template
3) translation: synthesis of proteins using messenger RNA as a template
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The three types of RNA
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1) Messenger RNA (mRNA): used to encode polypeptides (proteins)
2) Transfer RNA (tRNA)
3) Ribosomal RNA (rRNA)

Last two play part in protein synthesis but do not themselves encode genetic information for making proteins
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Three characteristics of Prokaryotic Genetics
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1) circular DNA
2) no membrane separating the chromosome and cytoplasm
3) vast majority have no introns
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Three characteristics of Eukaryotic Genetics
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1) linear DNA
2) chromosomes inside nucleus and ribosomes in the cytoplasm
3) all have introns between exons
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Primary Transcript
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both introns and exons transcribed into this an unprocessed RNA molecule that is the direct product of transcription
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Three characteristics of DNA structure
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1) DNA is a double-stranded molecule
2) forms a helical configuration
3) measured in terms of numbers of base pairs
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Nucleotide pairs (with number of hydrogen bonds)
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Adenine (A) pair with Thymines (T) [2 hydrogen bonds]
Guanines (G) pair with Cytosines (C) [3 hydrogen bonds]
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How do the complementary strands of DNA run in respect to one another?
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They run in an "antiparallel" fashion, where the sense strand (5' to 3') runs antiparallel to the antisense strand (3' to 5')
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Inverted Repeats
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Short repeated sequences in DNA molecules
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Melting/Denaturation
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breaks apart hydrogen bonds when two strands of a DNA molecule are separated by heat
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Annealing
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when denatured strands are cooled double stranded DNA molecule will reform
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Hybridization
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invovles putting together two DNA strands from different sources annealing can be used to do this
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Supercoiling
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very long DNA molecule can be packaged into the cell because it is further twisted can occur in a positive or negative direction.
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DNA gyrase
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produces negative supercoiling in bacteria and Archae belongs to group of enzymes caled "topoisomerases" classified as topoisomerase II
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Topoisomerase
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a group of enzymes DNA gyrase classified as type II of this
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Three facts about viruses
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1) Consist of either RNA or DNA
2) control their own replication
3) viral chromosome may be linear or circular
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Three facts about plasmids
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1) replicate separately from chromosomes
2) Double Stranded DNA that is usually circular but can be linear
3) Do not cause dammage and do not have extracellular forms
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Three facts about organellar genomes
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1) Examples are chloroplasts and mitochondria
2) DNA is separate from nuclear DNA
3) DNA replicated independently of nuclear DNA
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Transposable Elements
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Molecules of DNA can move from one site on a chromosome to another paly an important role in genetic variation
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DNA Polymerase
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Catalyzes the addition of nucleotides to the 3'-OH group
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Primer
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short (<15 bases) single stranded pieces of RNA primer strand synthesized by "primase" has an available free 3' OH
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Bidirectional replication in circular chromosomes
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1) replication is bidirectional from the point of origin of replication
2) two replication forks on chromosome replicating in opposite directions
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Exonucleases
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DNA Polymerase I and III have a proofreading function they can remove mismatched bases and replace them with the correct bases mutation rate 10^-8 to 10^-11 errors per base insertion
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Restriction Enzymes/Endonucleases (4 facts)
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1) Found in both Archae and Bacteria (rarely found in Eukaryotes)
2) recognize specific DNA sequences and cut the DNA at those sites
3) Essential for in vitro DNA manipulation
4) in prokaryotes protect them from invading foreign DNA
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How do prokaryotes protect their own DNA?
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They do so by modifying through methylation (specific bases within the recognition sequence have methyl groups attached to them)
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How does the Restriction Enzyme Digest work?
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DNA frament can undergo this Restriction enzyme will cut DNA at each site it recognizes eletrophoresis gel contains digested fragments and yields a restriction map
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What is nucleic acid hybridization and the southern blot used for and how do they work?
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used to check for the presence of DNA sequence in a DNA sampe Double stranded DNA fragments from a gel are transferred to a membrane and denatured Single stranded denatured DNA fragments are annealed to complementary strands probe containing a label or are probed using a labeled single stranded oligonucleotide
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What does DNA sequencing require? (5)
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1) DNA template
2) Primer
3) Deoxy nucleotides
4) Dideoxy nucleotides
5) A polymerase enzyme
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What method is preferred for DNA sequencing and what does this method entail?
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The Sanger method is used DNA is sequenced by making a copy of a single stranded DNA template strand polymerase adds nucleotides dideoxy analog acts as the specific chain termination reagent
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Polymerase Chain Reaction
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amplifys DNA multiplies DNA sequence by a billion fold
DNA template is dantured by heat (requires Taq polymerase, a thero-stable enzyme)
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Transcription Terminators (3)
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1) Intrinsic terminators that involve sequence patterns only
2) inverted repeats that lead to stem loop formation
3) GC rich followed by AT rich sequences
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Rho protein
answer
binds directly to RNA causes both the RNA polymerase and newly synthesized RNA strand to be released from the DNA
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Rho dependent termination site
answer
site where newly synthesized RNA is released from the DNA
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Operon
answer
Genes that are transcribed from a single promoter consitute this

clutster of genes arranged in a linear, consecutive fashion when a single regulatory protein controls a single operon
question
Codon
answer
triplet of bases that encode a specific amino acid 64 possible codons (4 bases taken three at a time 4^3)
question
Whats so special about the 3rd base position in the codon?
answer
1) changing this may not always change the amino acid
2) irregular base pairing may be tolerated at the 3rd base position
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Start codon
answer
AUG (begins the reading frame) establishes the open reading frame
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Stop codons (3)
answer
UAA, UAG, UGA (transcription terminates)
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Transfer RNA
answer
contain an anit-codon portion that base pairs with teh codon portion of the mRNA
question
aminoacyl-tRNA synthetases
answer
enzyme that helps tRNAs bind a specific amino acid
question
Structure of tRNAs (4 facts)
answer
1) short single stranded molecules with extensive secondary structure
2) have lengths of 73-93 nucleotides
3) have conserved and variable regions
4) have cloverleaf structure
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Anti-Codon
answer
one of the variable regions consists of three nucleotides found on the anti-codon loop
question
acceptor end
answer
where the amino acid is attached
question
how do antibiotics inhibit protein synthesis
answer
do so by inhibiting the activity of bacterial ribosomes
question
the four steps of protein synthesis
answer
1) Initiation: involves Shine Delgarno (located at the 5' end of mRNA binds by complementary base paring to regions in the 3' end of the 16S RNA portion of the ribosome sets up teh correct "reading frame")

2) Elongation
3) Termination
4) Release
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3 Key sites on the ribosome
answer
1) Acceptor Site: where the charged amino acid first arrives

2) Peptide Site: where the growing polypeptide chain is held

3) Exit Site: where the tRNA is released from the ribosome
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Polysome
answer
several ribosomes can simultaneously translate a single mRNA increases the speed of translation
question
Feedback inhibition
answer
occurs primarily in the regulation of entire biosynthetic pathways like in the biosynthesis of amino acids or nucleotides

1) the end product can regulate its own biosynthesis
2) the end product can inhibit the acitivity of the 1st enzyme in the pathway
3) shuts down entire biosynthetic pathway
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Allostery
answer
property that makes possible the process of feedback inhibition
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Active site
answer
where the substrate binds in an allosteric enzyme
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Allosteric Site
answer
where the inhibitor (effector) reversibly binds in an allosteric enzyme binding at this site changes the enzyme conformation
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Isozymes
answer
different proteins that catalyze the same reaction but are subject to different regulatory controls example is DAHP isozymes in E. coli
question
Covalent Modification of Enzymes
answer
a regulatory mechanism for changing the catalytic activity of an enzyme can be reversibly modified most times

Glutamine Synthetase is example of this, modified by addition of AMP (adenylation).
question
Protein Splicing
answer
occurs when amino acid residues are removed from the protein itself intein is removed (portion of the peptide that is removed)
question
Intein
answer
portion of the peptide that is removed during protein splicing are self splicing
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DNA Binding Proteins
answer
bind to DNA because of interactions betweeen specific domains of the proteins and specific regions of the DNA molecule most interact in a sequence specific manner major groove of DNA is an important site of protein binding
question
Protein-DNA interactions
answer
central to the completion and regulation of replication, transcription and translation
question
How are DNA molecules bound to proteins?
answer
amino acid side chains of the binding protein associates to the bases and sugar phosphate backbone of the DNA
question
How are DNA binding proteins classified?
answer
they are classified by structure or domains

1) Zinc Finger
2) Leucine Zipper
3) Helix- turn-helix
question
Positive control of transcription
Negative control of transcription
answer
-involves a regualtory protein (inducer) that activates the binding of RNA polymerase example is maltose catabolism in e.coli

-Enzymes that catalyze the synthesis of a specific product are not synthesized if the product is present in sufficent amounts
question
Inducer
answer
Substance that initites enzyme induction (ex lactose)
question
Corepressor
answer
substance that represses enzyme synthesis (ex arginine) effectively called effectors
question
Repressor proteins
answer
become active after it is bound by the effector block transcription
question
Operator
answer
region of DNA (near the promoter) where the repressor/effector complex binds single mRNA
question
Induction
answer
involves the binding of an inducer to the repressor instead of corepressor uses an inducer
question
Activator proteins
answer
bind to specific DNA sequences called activator binding sites activates transcription bind several hundred bases before promoter may bend the DNA
question
Regulon
answer
when more than one operon is under control of a single regulatory protein example is maltose catabolism can be involved with both poistive and negaitve control of transcription
question
Global Control System
answer
used by cell to make most efficient use of the available carbon sources cell always uses glucose first
question
Global Regulatory Systems
answer
regulate expression of many unrelated genes siumultaneously brought about by changes in the environment
question
Catabolite repression
answer
ensures that cells always use glucose first prevents the expression of all other catabolic operons affected by this control mechanism when cells are grown in a medium containing glucose
question
Glucose Effect
answer
1) best energy source is chosen
2) cell does not waste energy making enzymes for catabolizing the other sugars
3) if glucose is exhausted, next best sugar taken
question
Stringent Response
answer
a global translational control in prokaryotes that represses tRNA and rRNA synthesis during amino acid starvation

Triggered by alarmones, which inhibit rRNA and tRNA by interfering with RNA polymerase initions of transcription of genes.
1) guanosine tetraphophate ppGpp
2) Guanosine pentaphosphate (pppGpp)
question
Gene regulation
answer
can be brought about by changing the amount or activity of sigma factors, which recognize different promoter sequences (ex. e. coli has 7 different sigma factors each recognizing a different promoter)
question
How are sigma factors controlled?
answer
1) transcriptional controls (positive or negative)
2) Degradation by proteases (destroys, lowers concentration)
3) Anti-sigma factors (inactive sigma factors)
question
Sigma factors can also be used for these three different circumstances
answer
1) Nitrogen assimilation
2) iron transport
3) heat shock: produces heat shock proteins (sigma 32) under "normal" conditions sigma 32 is normally degraded very quickly following its synthesis and is more rare in the cell
question
Quorum Sensing
answer
sensing mechanism involving signaling molecules that allows Cells to survey their environment for cells of their own species.
Ensures that sufficient cell numbers of a given species are present before eliciting a particular biolgoical response.
Widespread in gram (-) bacteria.

Examples
1) V. Fischeri (bioluminescent ability)
2) P. Aeruginosa (produce a biofilm that can prevent antibioltic penetration)
3) S. Aureus (produce peptides that damange host cells and disrupt immune system)
question
Attenuation
answer
Mechanism whereby gene expression is controlled after the intiation of RNA synthesis only seen in prokaryotes.

Occurs because a portion of the newly formed mRNA folds into a unique stem-loop that causes cessation of RNA polymerase activity.
question
Signal Transduction
answer
When a cell receives an external signal through a sensor that then transmits the signal to the regulatory proteins.
question
Two components of regulatory systems
answer
1) Specific sensor kinase protein: aka histidine kinases responsible for self-phosphorylation; receive signal from the environment and phosphorylate one of their specific histdine residues.

2) Partner response regulator protein: DNA binding protein that regulates transcription phosphoryl group from histine kinase is transferred here.
question
Plaque assay
answer
Used to quantify the number of virions in a suspension accurate way to measure virus infectivity.

1) Bacterial culture infected with a virus and produces "lawn" growth in culture
2) virus will cause lysis of the bacteria it infects
3) lysed cells produce "clear areas" called plaques
question
Five stages of the life cycle of a virus
answer
1) Attachment
2) Penetration
3) Synthesis
4) Assembly
5) Release
question
Four stages of viral nucleic acid synthesis
answer
1) Eclipse: infectivity of the virus dissappears after adsorption of virus by host cell.

2) Latent period: replication of viral nucleic acid and protein occurs during this period.

3) Maturation: newly synthesized nucleic acid molecules are packaged inside protein coats.

4) Burst size: number of new virion particles that are released.
question
Three facts about viral attachment to the host cell
answer
1) attachment of a virion is a highly specific process
2) involves complementary receptors on the surface of a susceptible host cell and the infecting virus
3) if host cell receptor altered by mutation, the virus cannont adsorb and infect
question
Penetration of viruses
answer
Not the same in all virus-host cell interactions...
May become uncoated prior to entering the host cell
Entire virion may enter host cell and must become uncoated after entering the host cell
question
Restriction endonucleases
answer
An example of prokaryotic DNA destruction systems (because they lack immune systems)
Protect their own DNA with protective methyl groups at the restriction sites in their genomes
question
Genomes of T-even bacteriophages
answer
T2, T4, T6 are closely related viruses

T4 genome is most widely studied. it is a virulent virus (can kill cells through a lytic life cycle)
question
3 parts of T4 genome
answer
Early Proteins and Middle Proteins: primarily enzymes involved in DNA replication and transcription.

Late Proteins: head and tail proteins and the enzymes involved in liberating the mature phage particles from the cell
question
How does T4 appropriate the host cells RNA polymerase?
answer
1) may insert anti-sigma factor that causes the host cell RNA polymerase to not recognize host promoters-shuts down host transcription
2) may use its own proteins to covalently modify the sub-units of the host RNA polymerase
3) these modification cause the host RNA polymerase to recognize only phage promoters
question
Temperate Bacteriophages
answer
Viruses that do not neter a lytic life cycle
question
Lysogeny
answer
A stage entered into by temperate viruses.
Most virus genes are not expressed.
Virus genome (prophage) is replicated in synchrony with the host chromosome.
question
Bacteriophage Lamda
answer
A double stranded DNA temperate phage.
Lytic and lysogenic events in lambda controlled by several promoters and regulatory proteins.
Often infect E. coli.
Genetic switch controls whether a lytic or lysogenic pathway will ensue in bacteriophage lambda.
question
For lysogenic pathway to ensue it is necessary that (2)...
answer
1) production of all late proteins must be prevented
2) copy of the lambda genome must be integrated into the host chromosome
question
Three things animal viruses can cause:
answer
1) persistent infections: produce virus indefinitely cause lysis of host cell immediate lytic effect.

2) Transformation: coversion of a normal cell into a tumor cell.

3) Latent Infections: delay between infection by the virus and lytic events symptoms reappear sporadically.
question
Retrovirus
answer
Transfer information from RNA to DNA.
Contain a genome that is replicated through a DNA intermediate.
Uses an enzyme called reverse transcriptase to perform retro-transcription.
question
Viroids
answer
1) smallest known pathogens
2) do not encode any proteins-completely dependent upon host cell enzymes
3) cause some plant diseases (no viroid diseases in animals)
question
Structure of viroid (3)
answer
1) small, circular single stranded RNA molecules
2) contain no protein coat
3) genomes between 246-399 nucleotides
question
Prions
answer
1) cause a variety of disease in animals (scrapie in sheep, mad cow in cattle)
2) no known prion diseases in plants
3)Consist of protein, but have no nucleic acid.
4) Exist in native cellular form and pathogenic form, which replicates itself by converting native prion proteins into pathogenic conformation.
question
Structure of prion (2)
answer
1) have a distinct extracellular form that is entirely protein
2) do not contain any nucleic acid
question
Mutation
answer
an inherited change in a nucleotide base or base sequence in the DS DNA of the genome
Can be beneficial or harmful
question
Genetic recombination
answer
Genes from two separate genomes that are brought together.

A larger degree of genetic change occurs through recombination than through mutation.
question
Genetic Change in Prokaryotes
answer
Do not reproduce sexually
Prokaryotes can undergo genetic change due to gene transfer
question
Later Gene Flow
answer
Mechanisms that allow for gene transfer and recombination are based on this. Genes are transferred from donor to recipient horizontally rather than vertically from mother to daughter cell.
question
Selectable mutations
answer
Confer some type of an advantage to the organisms possessing them.

Example of this is drug resistance
question
Unselectable mutations
answer
No selective advantage is incurred.

Example of this is the loss of color in a pigmented organism.
question
Spontaneous Mutations can occur because of this...
answer
1) exposure to nautral forms of radiation
2) oxygen radicals
3) base pairing errors during DNA replication
question
Point Mutation
answer
If a mutaiton affects just a single base pair.
If point mutations are carried through transcription and translation they can end up altering the protein sequence.
Can have severe implications or can be unharmful.
question
Reversion
answer
point mutations may be reversed by this process

Same site revertants: site that is mutated that reverts back to the original base
Second Site revertants: second mutation occurs at a second site in the sequence compensates for the effect produced by the first mutation
question
Transition Changes
answer
Occur when a base mutation involves the substitution of one purine base for another purine base or one pyrimidine base for another
question
Transversion Changes
answer
purine for pyrimidine or vice versa
question
Base insertions
answer
mutations involving the addition of a base or bases
question
Base deletions
answer
mutations involving the deletion of a base or bases from the nucleotide sequence
question
Frameshift Mutations
answer
alter the reading frame of the mRNA by moving the reading frame up or down a single base or several bases
question
Mutations in RNA genomes
answer
Occur at a level that is 1000 fold higher than in DNA genomes.
There are no repair mechanisms but there are proof reading functions.
Leads to a heightened mutation rate in the RNA genomes of RNA viruses.
Makes RNA viruses able to evolve more rapidly.
question
Mutagenesis
answer
induced by agents called mutagens
question
Nucleotide base analogs
answer
Molecules that resemble DNA purine and pyrimidine bases in structure, yet display faulty base pairing properties.
Results in mismatched base pairs
question
Alkylating Agents
answer
React with amino, carboxyl, and hyroxyl groups in proteins and nucleic acids and substitute alkyl groups in their place.

Can induce changes even in nonreplicating DNA.
question
Radiation
answer
Both are used to create mutations.

1) ionizing- more powerful form of radiation than UV light because the ionization of compounds, which brings about the formation of potent chemical species such as free radicals. Has greater penetrating power than UV light and is potentially more destructive.
2) nonionizing: more widely used used in the form of UV light. Maximum absorption is at 260 nm.
question
Pyrimidine dimers
answer
One of the effects of UV light on DNA is the formation of this when two adjacent pyrimidine bases become covalently bonded to one another such that the probability of the DNA polymerase misreading the sequence is greatly increased.
question
Free radicals
answer
react with DNA causing "hits" to occur that result in mutations
question
Examples of short wavelength
answer
1) X-rays
2) Cosmic Rays
3) Gamma Rays
question
DNA Repair
answer
If an error in DNA synthesis is correct before the cell divides, no mutation occurs.
If DNA replication stalls, the cell will die.
question
SOS regulatory system
answer
An example of DNA repair process that has some aspects that occur in the absence of template instruction.
question
Hyperaccurate Phenotype
answer
Where an organism has an extremely efficient system of DNA fidelity repair and replication can be advantageous or disadvantageous
question
Advantageous
answer
when making repairs caused by exposure to radiation
question
Disadvantageous
answer
when it prevents an organism from evolving at a apce that is dictated by a changing environment
question
Homologous Recombination
answer
Genetic exchange between homologous DNA sequences from two different sources.
Involves crossing over.
Uses RecA protein.
Occurs only after the transfer when DNA from the host is in the recipient cell.
question
Transformation
answer
Occurs in Bacteria and some archaea process by which free DNA is incorporated into a recipient cell and bring about genetic change.
Occurs in B. subtilis fragmented DNA.
Only 1% of certain strains and species of prokaryotes are naturally competent (can be transformed)
question
incorporation of donor DNA into recipient cell
answer
Requires the activity of a single-stranded binding protein, RecA protein, and several other enzymes SS binding proteins prevent transforming DNA from degradation by nucleases
question
RecA
answer
Allows homologous recombination to occur which allows transforming DNA to be integrated into the genome of the recipient
question
Transduction
answer
Bacterial DNA is transferred from cell to cell by a bacterial virus.

Generalized transduction: virus incorporates random fragments of the host bacteria cell's chromosomal DNA into the viral genome. Efficiency is low.

Specialized transduction: DNA from a specific region of the host bactera chromosome is integrated indirectly into the virus genome and ususally replaces some of the virus genes. Efficiency may be very high. Only occurs in some temperate viruses.
question
Incompatible Plasmids
answer
if a plasmid is transferred to a cell that already carries another type of plasmid, the second plasmid may not be maintained and consequently lost during subsequent cell replication.
question
Conjugation
answer
Involves cell to cell contact. A replicative process whereby both cells end up with copies of the plasmid. Process of genetic transfer.

Sometimes other genetic elements such as all or parts of the host chromosome can be transferred during conjugation.
DNA replication must take place during conjugation such that both the donor and recipient cells have fully formed plasmids at the conclusion of conjugation.
question
Conjugative Cells
answer
Plasmids that govern their own transfer by cell to cell contact. Not all plasmids are conjugative.
question
Resistance Plasmids
answer
Known as R plasmids, among the most widespread and well-studied groups of plasmids, confer resistance to antibiotics and other growth inhibitors.
Resistance is spread between bacteria through conjugation.
question
Colonziation factor antigen
answer
Allows certain bacterial pathogens to colonzie the small intestine prior to secreting endotoxins.
question
Rolling Circle Method
answer
Method of DNA replication during conjugation
question
Episome
answer
it can integrate into the host chromosome
question
F Plasmid
answer
Found in E. coli

Mobilization: When integrated into the chromosome, the chromosome becomes mobilized. This can lead to a transfer of chromosomal genes to a recipient cell.

F+ Plasmids: Cells that have an unintegrated F plasmid.
Hfr Plasmids: High frequency recombination F plasmid integrated into the chromosome.
F' Plasmids: previously integrated F plasmids that have deintegrated and excised some chromosomal genes.
question
Insertion Sequence (IS)
answer
Integration of the F plasmid into the host chromosome occurs at this site.
These sites are regions of DNA sequence homology between chromosomal and F plasmid DNA.
question
Complementation
answer
recombination takes place between the donor chromosome and the recipient chromosome
question
Gene Cloning
answer
Involves molecular manipulation in vitro of vectors using PCR, RE, synthesizing DNA probes, DNA sequencing, gel electrophoresis etc. purpose is to isolate multiple copies of specific genes or regions of a chromosome in pure form.

1) isolating a gene of interest onto a fragment of DNA
2) experimentally control the replication
3) amplify copies
question
How are plasmids used as cloning vectors?
answer
1) they are easy to isolate and purify
2) can multiply to high copy numbers in bacterial cells
3) presence of selectable markers like antibiotic resistant genes
question
Plasmid pBR322
answer
Replicates in e. coli, small genome size can be maintained stably in its host, can by amplified to very high copy numbers, reasonable amount of foreign DNA can be inserted into it.
question
Insertional Activation
answer
Foreign DNA is inserted into one of these restriction sites contained within an antibiotic resistant gene, antibiotic resistance will be lost.
Can be used as a means of selecting transformed bacterial clones that have picked up the insert.
question
Second Generation Plasmid Vectors
answer
Higher utility and even easier to use than pBR322

Example is pUC19, which contains a multiple cloning site polylinker and is contained within the coding region of the gene that encodes B galactosidase

Blue colonies
bacterial clones that contain pUC19 without the DNA insert (B galactosidase is not disrupted and remains active)

White colonies
contain pUC19 with the DNA insert (B galactosidase is disrupted and inactivated)
question
Use of site directed mutagenesis
answer
Comparison of wild type protein versus a mutated protein. Examine the efficiency of proteins and/or enzymes with a particualr amino acid substitution.
Examine how mutation of a gene promoter affects transcription.
question
Bacteriophage Lambda as a cloning vector
answer
1) isolate vector DNA from phage particles and digest with the appropriate RE
2) connect the two lambda fragments with the fragments of foreign DNA using DNA ligase
3) packaging of DNA insert by adding cell extracts containing the phage head and tail proteins and allowing viable phage particles to form
4) transfecting E. coli and isolatin...
question
Why are bacteriophages like lambda advantageous?
answer
1) transfection rate is greater than the transformation rate of plasmid vectors
2) can hold larger amounts of DNA than most plasmid vectors
question
Antigenic shift
answer
A phenomenon in which portions of the RNA genome from two genetically distinct strains of virus infecting the same cell are reassorted.
question
Antigenic drift
answer
When the structure of the neuraminidase and hemagglutinin proteins on the surface of the influenza virus virion is altered, usually in subtle ways, by mutation in the genes encoding them,
question
Plant viruses
answer
(+) strand RNA genomes
Tobacco Mosaic Virus, first discovered
Can move within the plant through intracellular connections that span the cell wall
question
Animal viruses
answer
(+) strand RNA genomes
Poliovirus
Rhinovirus
Coronavirus
Hepatitis A

The viral RNA is translated directly, producing a long polyprotein that is broken down by enzymes into the numerous small proteins necessary for nucleic acid replication and virus assembly.
question
(-) Strand RNA Viruses
answer
The virus RNA is not the mRINA, but is copied into mRNA by an enzyme present in the virion.

Rabies virus
Influenza virus
question
Bacteriophage
answer
virus the infects prokaryotic cells
question
Reoviruses
answer
Contain segmented linear double-stranded RNA genomes.
Contain an RNA-dependent RNA polymerase within the virion.
question
Retroviruses
answer
Contain RNA genomes and use reverse transcriptase to make a DNA copy during their life cycle.
question
Hepadnaviruses
answer
Has a DNA genome which replicates by way of an RNA intermediate.
question
Replicative form
answer
a double stranded DNA or RNA molecule that is an intermediate in the replication of viruses with single stranded genomes.
question
Transposase
answer
an enzyme that catalyzes the insertion of DNA segments into other DNA molecules
question
Reverse transcriptase
answer
The process of copying genetic information found in RNA into DNA.
question
RNA replicase
answer
an enzyme that can produce RNA from an RNA template.
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