MMG 301 Exam 3 Flashcards

DNA
Terms Definitions
- silent mutation:
change in the DNA sequence, but the new codon codes for the same
amino acid
non sense mutation
results in a STOP codon
missense mutation
results in a different amino acid
- frameshift mutation
results from an insertion or deletion that changes the triplet
reading frame of the mRNA
spontaneous mutation
occur on their own
- Causes of Mutations
- chemicals (base pair analogs, alkylating agents)
- UV, ionizing radiation
- errors in DNA repair and replication
- reversions
point mutation giving restoration of a wild type phenotype
- same site
base pair mutates back to original genotype
- second site
mutation at a different site restores the wild type phenotype
- EX: a base pair insertion that corrects an altered reading frame cause by a nearby
Deletion
- Brenner and Crick used frameshift mutants to demonstrate that 2 bases of
DNA encode 1 amino acid
- insertions
new stretches of DNA are added
- translocations
rearrangements of regions of DNA; large segments of DNA move to another location on the genome
- inversions
orientation of a segment of DNA is reversed, sometimes large portions of genomes involved
auxotroph
a mutant with a nutritional requirement for growth
How to isolate nutritional auxotrophs
1. master plate contains colonies – unmutated and possible mutated cells
2. cells of each colony are transferred to velveteen cloth
3. cells from velveteen are then transferred to several plates (complete agar medium and
minimal agar medium)
mutagens:
substances that cause mutations; often chemical compounds that react with or bind
to DNA
- Ames Test
1. Expose suspected mutagen to live enzyme extract
2. Place resulting chemicals on filter disk
3. Spread bacteria onto plate made with agar medium without histidine
4. Place filter disk on plate
5. Grow plate overnight and examine for colonies
6. By using proper controls, an increase in the number of colonies near the filter disk indicates that the chemicals (or the enzymatic products after metabolism by the liver enzymes) indicates that the tested compound is a potential carcinogen
a. More colonies on plate
- homologous recombination
: exchange of DNA segments from one DNA molecule to another
- Genetic exchange and Recombination in Bacteria
1. Nick: one of DNA molecules cut of one DNA strands in double helix
2. SBB: nicked strand detaches from other strand with help of proteins, especially single
stranded binding protein
3. strand displacement: nicked strand displaces homologous regions of recipient DNA
4. crossed-strand exchange: formation of a crossed strand structure (Holiday junction)
5. resolution: cutting of DNA strands to yield new DNA with heteroduplex regions:
segments orienting from different DNA molecules
- transformation
incorporation of free DNA into recipient cell
- natural competence
only certain microbes have the natural ability to take up foreign
DNA (haemophilus, streptococcus, bacillus)
- artificially induced competence
induced to uptake plasmids by exposing them to
certain growth condtions followed by chemical treatments
- e. coli cells are suspended to promote transformation by plasmid
- electroporation
- transduction
bacterial virus mediated transfer of DNA
- generalized: random pieces of chromosomal DNA are transferred by a bacterial virus
(phage) particle
- specialized: chromosomal segments adjacent to integrated phage DNA are transferred
- only occurs are certain sites in the chromosome
conjugation:
direct plasmid transfer or integrated plasmid-mediated chromosome transfer
- Interrupted Mapping
1. mix cells
2. incubate – remove aliquots and 10 min time points
3. mix to separating mating cells
4. plate on selective media
- medrodiploid
two copies of a gene within a single cell
- second copy of gene carried on a transducing phage
- second copy of gene carried on F plasmid
- complementation
second, normal copy of a gene in a single cell can fulfill the role of the
original mutated gene, thus restoring the cell to the original phenotype
- mutant gene and normal gene are located on different DNA molecules
- no recombination involved
- transposition
movement of genes from 1 location in a genome to another
- rare event
- mediated by transposable elements
- insertion sequences: simplest, encode a transposase gene with flanking
inverted repeats
- operon
: contiguous set of gene transcribed as a single mRNA under the control of a single
regulatory region
- regulon
spatially separated genes regulated by a common regulatory molecule (globular
regulation)
Regulation of biochemical pathways can occur at several points
- enzyme activity: enzyme is altered by a regulatory molecule
- rapid regulation, but why expend more energy to make an enzyme you don’t need
- amounts of enzymes
- transcriptional regulation: control of synthesis of mRNA
- control of translation
- slower but potentially more energy efficient method
Allosteric regulation
- regulation of activity by enzyme inhibition – often found in enzymes involved in biochemical
pathways
- as levels of the end product of a pathway build up, the first enzyme is inhibited
- feedback inhibition
end product represses enzyme activity of first enzyme
1. effector molecule binds to enzyme and alters the conformation
2. substrate can no longer bind and react in the same active site
-Transcription
- promoter: specific site upstream of a gene where RNA polymerase binds
- sigma factor: recognizes specific DNA promoter site; aids binding of RNA polymerase; is \ released as transcription begins
- only 1 strand is transcribed
- mRNA is produced as RNA polymerase
- moves 5’ to 3’ along the DNA
- transcriptional control: different sigma factors are used for simultaneous regulation of many
genes (global regulation)
- alpha 32: synthesized when cells face a sudden shift in growth temp or heat shock
- recognizes and binds to 36 genes the protect the cell
- alpha 54: made when cells are grown without added ammonia or “nitrogen starvation”
-turns on transcription of 12 genes that allow growth on other N sources
-alpha 38: survival is stationary phase
- regulon: use of an alternative sigma factors allow for simultaneous control of multiple
genes/operons
- each of these factors recognizes a distinct promoter sequence
- repressor: dimeric protein binds to symmetric inverted repeat sequences on DNA
-Repression (negative control)
- arginine operon of E. coli: add arginine to medium of growing cells, genes for arginine synthesis
shut down
- transcriptional regulation
- arginine binds to free repressor protein
- arginine-repressor protein complex can then bind to operator
- no arginine -> make genes to produce arginine
- arginine present -> repress genes to produce arginine
- Induction (negative control)
- de-repression
- lac operon of E. Coli: add lactose to medium of growing cells, genes for lactose utilization turn
on
- transcriptional regulation
- inducer binds to operator-bound repressor protein
- repressor protein released from operator site
- utilization of lactose as a carbon source
- lactose present -> induce genes to utilize lactose
- Induction (positive control)
- maltose operon: activator protein bunds inducer (maltose
- genes for maltose utilization are transcribed
- many genes don’t require an inducer
- some activator binding sites can be very far from promoter
- Signal Transduction
the process of transmitting external signals relevant to regulatory target
- sensor kinase protein
- response regulator protein
1. sensor kinase binds signal molecule and phosphorylates itself
2. phosphate is transferred to the response regulator molecule
3. phosphorylated response regulator then binds to operator on DNA
- catabolite repression
if a more favorable carbon source (glucose) is available, then genes for
utilization of other carbon sources (lactose) are repressed
- diauxic growth
cells grow on the referred growth substrate and then switch to the second, less
preferred substrate
- multiple C sources, use one until gone, then use next one
-Control of translation
- antisense RNA: can be transcribed from distant genes
- short and complementary with 5’ end of gene A = no translation
- riboswitches: mRNA changes conformation in presence of metabolite; prevents translation
- stringent response
happens in a nutrient shift down when transferring from rich to minimal
1. as internal pool of AA is depleted, some ribosomes will contain bound uncharged
tRNA
2. results in production of ppGpp and pppGpp by the ribosome bound RelA protein
3. production of tRNA, rRNA, and ribosomes is decreased, and synthesis of amino acid
biosynthetic pathways are activated
- ppGpp is termed alarmone because it signals that AA starvation is occurring
Preparation and isolation of DNA fragments
- joining DNA from different origins yields recombinant DNA
- a large population of heterogeneous fragments (called a library) can be joined in a singly type
of cloning vector
- need restriction enzymes (endonucleases)
- recognize a 4-8 bp sequence in dsDNA
- make a cute in both strands of the DNA
- staggered: sticky ends
- blunt
- need gel electrophoresis to separate fragments
- agarose gel
- need DNA ligase
- Joining DNA fragments into cloning vectors with DNA ligase
- sticky ends base pairing stabilizes joining and facilitates ligation
- Cloning of DNA fragments from genomic DNA into plasmid vectors
- want to get all genomic DNA fragments cloned into plasmids
- need lots of pasmid clones
- can select for desired clone
- PCR
- cycle repeated 12 to 20 times
- sequencing/ evolutionary relationships
- diagnostics
- detection of viable but non culturable microbes
- denaturation 95 C
- annealing of primers with DNA 55 C
- polymerization 73 C
Cloning Vectors
- plasmids: pBR322 – one of the first
- ampicillin (beta lactam) resistance: used to select only for cells that contain plasmid
- origin of replication
- select for insertion of fragments using antibiotic genes
- cosmids: plasmid containing the cos site of lambda
- contains genetic elements required for packaing DNA into lambda particles
- can clone large segments of DNA
- use an in vitro packaging extract
- artificial chromosomes
- yeast: propogate like a separatechromosome; large DNA inserts
- bacterial: based on E. Coli F plasmid; inserts up tp 300 kbp
- shuttle vector: has origin of replication for eukaryotic organism and origin of replication for e. coli
- Application of recombinant DNA technology
- shotgun approach
- fragment the DNA
- clone fragments into plasmids
- transform e. coli cells and grow in liquid culture
- purify plasmid DNA and sequence the inserted DNA
- top side down sequencing
- start with large, ordered clones of the DNA to be sequenced (mapped to their location
on the DNA
- each ordered clone is then further fragmented and sequenced
- pyrosequencing: 400 million base/10 hr run
metagenomics
- community sampling: PCR amplify 16s rRNA genes and then sequence
- total community sequencing: shotgun cloning and sequencing of total genomic DNA isolated
from whole microbial communities
- compter processing of data
- can produce complete or nearly complete genomes of thousands of uncultured
microorganisms
-human skin and intestinal tract
- Sargasso sea
- acid mine biofilms
- marine sediments
- soil samples
- E. Coli Genome
- determined in 1997
- single, circular molecule
- open reading fragments: appear to be able to code for proteins
- 38% unknown function
- Streptococcus pyogenes
- causes toxic schock syndrome
- flesh eating bacteria
- larger than other strep genomes
- similar to bacteriophage
- DNA Microarrays
use 1 microbe grown under 2 separate conditions
- isolate mRNA and convert to DNA
- tag with fluorescent tag
- hybridize on chip and analyze
-*chip contains short complementary DNA fragments
- each tag is unique to each gene
- hybridize: allow to base pair
- allow to hybridize with oligonucleotides on gene chip
- use laser to excite tagged molecules
- can detect changes in mRNA levels for thousands of genes in a single experiment
- Proteomics
- simultaneously examining regulation og all of the proteins in the cell = the proteome
- cell proteins are separated into 2 dimensions
- spot patterns are compared for cells from cultures grown under different conditions
- protein spots can be examined by mass spectrometry and IDed by molecular weights
- Commodities
- food additives: amino acids, thickening agents, vitamins
- solvents: butanol, ethanol
- biofuels: ethanol, methane, hydrogen
- processes
- sewage treatment
- bioleaching: extraction of metals from ore
- bioctalysis: production of chrial compounds
- fine chemicals
- pharmaceuticals: antibiotics, antifungals
- lab and diagnostic reagents: enzymes, proteins
emerging technologies
- DNA and protein array technology
- biosensors
- fermentation
large-scale growth of microorganisms
- process control: mixing, aeration, O2 monitoring, temperature control, sterilization, pH
- lab scale: 1-10 liters
- pilot plant scale: 100-3000 liters
- commercial scale: up to 500,000 liters
- air lift fermentors: air flow keeps culture mixed
- fixed-bed reactor: microbes grow on pourous solid surface
- solid state: growth without added water
1. Grow starter cluture in liquid medium
2. sterilize solid growth substrate with steam
3. inoculate substrate
4. grow in uniform layers in sterilized trays
- fluidized-bed reactor: microbes grow on surface of inert particles suspended in flowing
growth medium
- primary metabolites: substances produced during the primary growth phase of a culture
- secondary metabolites: produced during stationary phase
- unpredictable, non-essential for growth, specific conditions
- streptomyces
many of these bacteria behave like fungi and produce antibiotics
- Strain Optimization
1. spore-forming Gram+ bacteria
2. actinomycetes (streptomyces)
3. fungi
- Gentic Strain Optimization
- regulatory mutants: select mutants that have lost normal feedback inhibition of biosynthetic
pathway
- altered cell membrane to allow for secretion of amino acids product
- Optimization of Growth Conditions
add different precursurs during fermentation
- Metabolic pathway engineering
- genetic alterations for addition or mutation of specific steps in pathway designed to enhance
the production of desired substances
- incorporation of genes from other organisms adds enzymes
- Biocatalysis
- use whole microbial cells as live “bag of enzymes:
- production of cortisone
- grow in large fermentor, add precursure compound, purify product, complete
cortisone production
- Tetracyclines
- 300 genes involved in tetracycline production in streptomyces aureofaciens
- Riboflavins
- biologicial production is now replacing chemical synthetic methods
- Wine
- must: juice from crushed grapes
- pomace: skins, stems that are left in the must during fermentation, release tannins
- Beer
- malt: germinated barley seeds, rich in enzymes that break down starches into maltose
- mashing: mixture of malt and grains are cooked and steeped
- starches are digested, producing wort
-Copper Ore
-copper exists as sulfides
- Cu1+ is insoluble
- insoluble forms converted to sulfate salts by leaching with dilute sulfuric acid
- if an ore has low abundance of copper, then microbial leaching can increase yields
- Bioleaching
- use of microorganisms for recovery of metal sulfides from ore using acidithiobacillus
ferroxidans or leptospirillum ferrooxidans
- also used for extraction of uranium and gold
- need acidic pH
- Microbial mercury resistance
- Hg0: elemental mercury; liquid metal at room temp
- Hg2+: mercuric ion; forms soluble salts
-methylmercury: Hg2+ readily accepts methyl groups through microbial activity
- very toxic
-mercuric ion reductase system
1. mercury binds to MerP protein in periplasm
2. membrane bound MerT protein transports Hg2+ into cytoplasm
3. Hg2+ is substrate for mercuric ion reductase (MerA) and is reduced to Hg0
- Hg0 is volatile and evaporates
- undesired microbial degradation problems
- in storage tanks, microbes need water, so organisms grow at oil-water interfaces under
anoxic conditions hydrogen sulfate is produced, which is corrosive
- biofilms can polug rock pores and reduce fluid flow in petroleum recovery
beneficial actions of microbes
aid in petroleum recovery
- production of gasses in iol formations to increase reservoir pressure
- production of extracellular surfactants that decrease water surface tension in
oil deposits
- bioremediation of petroleum spills
- metabolize and oxidize to CO2
- application of petrophillic microbial suspension
- application of growth-promoting substances
- Microbial degradation of xenobiotics
- xenobiotics: synthetic chemical compound that does not naturally occur in nature
- complete degradation can occur in a single organism or by cometabolism
- combined metabolic activity of 2 or more different organisms
- mineralization: complete conversion of a xenobiotic compound into small inorganic products
-Dehalogenation
- critical ability for microbes to metabolize many xenobiotics
- reductive dehalogenation: halogenated organic compounds are used as terminal electron
acceptors
- most compounds found in anoxic subsurface environments
- aerobic dehalogenation: involves O2, often using the compound as a growth substrate
- usually initiated by oxygenase enzyme followed by multistep degradation of the
Compound
In-situ bioremediation
- growth of contaminant degrading microbes is promoted by injection of food sources or
addition of live cultures directly onto contaminated site
- C and N sources are injected into subsurface and metabolized products are pumped out by
extraction wells
- oxic
outside of aggregate, bathed in oxygen
- microoxic:
less oxygen, middle of aggregate
- anoxic
no oxygen, center of aggregate
- species richness:
number of populations or species
- species abundance
number of individuals within a population
- Cultivation Independent Methods
- microscopy
- Center for Microbial Ecology Image Analysis System
- snapshot image of organisms to see diversity
- DAPI/ Acridine Orange
- dyes that distinguish things that contain DNA
- LIVE/ DEAD
- dead cells dyed red because their cell wall is permeable
- Flourescent antibodies
- specific species or gene can become fluorescent if they contain
- GFP
- green fluorescent protein gene can be attached to certain area and pit back in
its normal region to track
- FISH
- phylochips
- PCR
- clone libraries
- shotgun sequencing
- 454 sequencing
- denaturing gradient gel electrophoresis
- terminal restriction fragment length polymorphism
- exponentially increases amount of DNA everytime it is run through the cycle
- amplified DNA = amplified fluorescence
- allochthonous
organic matter that enters the ecosystem from “outside”
- guild
group of organisms performing the same metabolic function
- Sulfur Cycle
- sulfur oxidation: can be aerobic or anerobic
- sulfate reduction: SO4 2- to H2S
- sulfur reduction: S0 to H2S
- sulfur disproportionation: S2O3 2- to H2S
-phyto plankton
floating oxygenic phototrophs
- benthic species
phototrophs attached to the bottom or sides of a body of water
- cyanobacteria
- algae
- guild: perform the same metabolic function
- primary producers
- oxygen
- much of organic matter synthesized by phytoplankton during photosynthesis is released as
dissolved organic matter (DOM)
- DOM consumed by bacterioplankton which becomes part of the suspended particulate organic
matter (POM) pool
- bacteria then consumed as food by protozoa predators
- some assimilated nutrients in bacteria and protozoa are mineralized and then assimilated
directly by phytoplankton without transfer to higher trophic levels in the ecosystem: microbial
loop
- oligotrophic lakes
- nutrient poor
- low microbial populations
- O2 rich
- eutrophic lakes
- nutrient inputs from outside
- high microbial populations
- O2 poor
- Biochemical Oxygen Demand
- microbial oxygen capacity of a body of water
- rivers are well mixed because of flow turbulence
- some parts may have low O2 levels due to microbial respiration
- input of sewage or organic waste
1. rise in bacterial activity
2. resulting decrease in dissolved O2
3. NH4+ rises from sewage input but then falls due to nitrifying bacteria
4. photosynthetic algae and cyanobacteria growth increases O2
- thus, self-purification process that results in oligotrophic conditions and
replenished O2 levels
osmiophiles:
require salinity and ions of seawater for cultivation
- psychrophiles
hypertonic; 5 C temp
-barophiles
in deep sea with great hydrostatic pressure
- oligocarbophiles
adapted to low concentration of organic C in ocean seawater
- Prochlorphytes
prokaryotic phototrophs that are phylogenetically related to cynaobacteria
- trichodesmium: N-fixing
- ostreococcus: small, eukaryotic, photosynthetic
- proteorhodopsin
light sensitive pigment that may be used by Pelagibacter to convert light
energy to ATP
- Treatment of Sewage
- primary treatment: removal of insoluble particulate materials from raw sewage by screening
and gravitational settling in tanks to make sludge
- physical separation
- secondary anoxic and aerobic treatment: microbial conversion of organic matter into microbial
biomass and final decomposition products
- aerobic human waste
- turn complex carbons into CO2
- lower Organic Matter
- trickling
- activated sludge
- slime results in flocculent material that serves as growth stratum for
protozoans and other organisms
- anaerobic: farming
- covered vessel used
- decompose to methane to be used as power source
- waste from food ad dairy
- tertiary treatment: biological and chemical removal of inorganic nutrients to reduce
eutrophiciation, virus removal, trace chemical removal
- effluent water is end result
- coliforms
gram negative, non-spore forming, rod-shaped bacterial that ferment lactose with
gas formation
- Wood
- wood contains cellulose, lignin, and hemicelluloses
- low N content
- mutual symbiosis: termite completely dependent on gut microflora to provide its C+N –
nutrition derived from anaerobic degradation of the wood particles
- cellulases: enzymes that degrade cellulose, are produced by the termite, by cellulolytic
protozoans symbioonts, or by ingested cellulose containing fungi
- certain spirochetes are also found that fix N2 that the termine uses as a nitrogen source
- lower termites: populations of protozoans and other microbes in the guy anaerobically
degrade cellulose to acetate (which is absorbed into the termite) and Co2 and H2
- higher termites: use social behavior to cultivate and ingest cellulose producing fungi
- Ruminant Microbe Symbiosis
1. food enters rumen where microbes begin anaerobic cellulose digestion
2. mixture goes to omasum and concentrated by water absorption
3. goes to abomasums (true stomach); acid kills and digests microbes
4. next to small intestine for further absorption of nutrients
- celluloytic bacteria and protozoa extracellulary hydrolyze cellulose to cellibiosone and
glucan oligosaccharaides
- decomposes plant material into volatile fatty acids that are absorbed and utilized by the animal, plus gas fermentation products that the animal expels
- acetate, propionate, butyrate
- Wolbachia – insect symbiosis
- infects developing sex cells of its host and gets passed from mother to child in egg
- when a male is infected, won’t be found in sperm
- bacteria that end up in the males produce a toxin that alters sperm
- tainted sperm cant fertilize eggs
- infected females can produce an antidote to restore sperm viability
- in wasps, wolbachia alters sex life of females so they no longer need a male to reproduce
- Riftia bacterial symbiosis
- large tube worms growing near deep ocean hydrothermal vents
- bacteria live in trophosomes
- Buchnera and Pea Aphids symbiosis
- food source is low in amino acids
- bacteria live in specialized cells called bacteriocytes which are surrounded by the host derived
membrane
- insect provides energy, C and N sources
- Microbe-bird symbiosis
- African honeyguide lives on beeswax, intestines contain microbes to digest wax
- hoatzin: leaves are major food source

-- contain a crop where bacteria aid digestion of cellulose
- Newcastle Disease
poultry
- transmitted though infected birds droppiongs and secretion from the nose, mouth and
eyes
- some strains are mild
- West Nile Virus
birds, horses, humans
- insect vector is mosquito
- weakness, ataxia (uncoordinated muscles movement),
- horse vaccine available
- Equine Infectious Anemia
horses
- insect vector is mosquito
- weakens immune system
- red blood cell formation impaired
Rabies
many mammals, esp bats
- attacks nervous system
- dumb rabies: animal becomes depressed and lethargic
- furious rabies: attack randomly and harm itself
- Chronic wasting disease
deer and elk
- neurological
- caused by prions
- Avian Influenza
- H5N1
- different subtypes due to changes in proteins
- ssRNA enveloped viruses
- 229 cases, 131 deaths
- antigen: recognized by immune system
- 5 present
- NP is a type specific antigen (A, B, C) which provides classification of human flu
- 16 hemagglutin subtypes (H1-H16)
- 9 neuramidase subtypes (N1-N9)
- use antibody test to determine which type
- A: occurs in pigs, birds, horses, and humans
- C: dogs, pigs, or humans
-B: Humans only
- Salmonellosis:
birds, animals, humans
- spread by ingestion or direct contact with contaminated poultry eggs, dariy, meat
- infects gut of animal and causes diarrheal illness
- Bovine Tuberculosis
- from mycobacterium bovis or mycobacterium tuberculosis
- Type C Botulism
- animal ingests spores during feeing on aquatic shores
- clostridium cotulinum
- Type E botulism
- ingested by mussels, fish, birds
- causes paralysis and death
- Lyme disease
- caused by borrelia burgdorferi
- begins as rash but spreads
- fever, lameness
- can lead to paralysis
- spread by ticks
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