Lecture Exam 3 Flashcards
Bacterial Transformation
Uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell’s genome
Transformation experiment by Fred Griffith in 1928
Infecting mice with Streptococcus pneumoniae, extremely pathogenetic to mice
- injection of mice with heat-killed pathogenic S and live nonpathogenic R strains (mice killed)
- Analyses of bacteria in mouse blood showed living S
- Concluded that living R has been TRANSFORMED to living S
Expain the differene between the R and S strain.
Pathogenicity requires polysaccharide capusle
S = smooth colony formers, make capsule
R = Rough colony formers, no capsule
* Arise in cultures of S, do not revert
* Concluded to be mutants affected in capsule formation
Griffith’s additional expirments
- Mouse is not essential for trasformation process, can preincubate heat-killed S with living R and obtain same result
- Cultivating living R in the presence of S cell-free extract produces same result
- Set the stage for fractionation experiments to obtain the transforming substance
Avery, Macleod and McCarty’s Expirment in 1944
- Removed proteins, lipids, polysaccharides, ribonucleic acid from extract by chemical and/or exymatic methods
– No effect on transformation - Purified DNA transformed
– Concluded DNA was transforming principle - Could purify DNA from R taht was transformed to S, and use that DNA to transform naive R strain
– Concluded that DNA was the hereditary material
Results met with surprise and disbelief
Wilson, 1925, in The Cell in Development and Inheritance’s new understanding
The differences ebtween differnt chromosomes depends on their protein components and no upon their nucleic acids
Objections to the Avery, McCarty, and MacLeod results
- Trace amounts of protein beyond the limits of detection were co-purifying with DNA, and this protein was the gentic material
- Exeriments showing DNase but not protease removed activity should have overcome this objection
– opponents argued that DNA was important scaffold but the important protein was protease-resistant - While amazingly pure preparations were made, only other evidence for DNA as hereditary material in the 1950s overcame these objections
Antibiotic Resistance
No special case scenario, 1950s
- Hotchkiss isolates penicillin-resistant and streptomycin-resistant S. pneumoniae
– DNA from each of these can be used to transform senstitive strains - The ability to trasnform cells to abitbiotic resistance also opened the way to analyses of the efficiency of transformation
– Led to examination of conditions for cometence
Hershey and Chase, 1952
DNA as Genetic Material
- Used bacteriophage T2 infection as model
- DNA labled with 32P; protein coat labled with 35S
- Only DNA entered cell but both new DNA and protein coats synthesized and incorporated ito new viruses indicating that DNA had the gentic information for synthesis of both of these viral components
Quantitative analyses of transformation and competence
- Addition of different amounts of DNA, and the pre-incubation of cells with DNA that could not confer drug-resistance, indicated that there were “receptors” for DNA on the cell, and that the receptors were saturable
- Sychronization of S. pneumoniae cell division by temperature shift protocols indicated that the organism was differentially competent for transformation at different stages of the cell cycle (1954)
Bacterial Cell Transformation Steps
- Binding DNA
- Uptake of single-standed DNA
- RecA-mediated homologous recombination
Bacillus trasformation, 1958
Because it was a more tractable system, became favored over S. pneumoniae
E. coli trasnformation, 1970
- Treatment of cells with calcium chloride necessary for transformation
- Electroporation has become the more favored method for transforming E. coli
Bacterial Plasmids
- Small, autonomously replicating DNA molecules that can exist independently or, as espisomes, integrate reversibly into the host chromosome
- Conjugative plasmids such as the F plasmid can transder copies of themselves to other bacteria during conjugation
What restriction enzymes recongize 6 nucleotides, 4?
Recognize 6
* EcroRI
* Pstl
* Smal
Recognize 4
* Haelll
* Hpall
5’ overhang
5’ -G-A-A-T-T-C
-G 5’ A-A-T-T-C-
Restriction enzymes that cleave the DNA asymmetrically leave several si
3’ overhang
5’ - C-T-G-C-A-G-
-C-T-G-C-A 3’ G-
Restriction enzymes that cleave the DNA asymmetrically leave single-stra
Blunt Ends
5’ - G-G-C-C-
-G-G 5’ C-C-
What make up a Plasmid?
- lacZ(alpha)
- Amp^R
- ori
what are teh 4 mech?
Horizontal (lateral) gene trasnfer in prokaryotes
transformation, transduction, conjugation, and via gene transfer agents
* genes can be transferred to the same or different species
* Trasnder of genes donor to reipient
- Exogenote
- endogenote
- Merozygote
Exogenote
DNA that is transferred to recipient
Endogenote
genome of recipient
Merozygote
recipient cell that is partially diploid as result of trasnfer process
Bacterial conjugation
DNA is transferred from a donor to a recipient bacterium by direct contact
* Direct cell to cell contact mediated by the F pilus
* A type IV secretion system
Who/what are the men that discovered bacterial conjugation?
- Transfer of DNA by direct cell to cell contact by Lederbuerg and Tatum, 1946
- Beadle and Tatum earned Novel prize for theory of one gene/one enzyme
- Davis shows in 1950 that direct contact is essential for transfer of genetic information
- Hayes (1952) shows gene trasnfer in polar (nonreciprocal)
- Joshua Lederberg adapted the reasoning used by Salvadore Luria and MAx Delbruck in their discovery of the jackpot nature of mutation to examine gentic recombination
- Specifically, Lederberg devised methods to look at low frequency events taht woul dbe evidence for gentic recomination
- He first had to show that bacterial life cycles had a dexual state- that they mated
Auxotrophs
- Originally identified becuase of requirement for nutritional source present in complete medium but not minimal medium
- Derived from aux- (Gr., increase) and trophe (food)
Prototroph
- Originally identified on basis of growth in minimal medium
- Deprived from protos (Gr., first/minimal) and trophe
Auxotrophs reverted to prototrophy
- Many of the E. coli auxotrophs reverted to prototrophy with a low frequency
- Approximately 1 in 10^6 cells
- Such reverse-mutation would frustrate analysis of low frequency recombination
- Lederberg conceived idea of using multiple auxotrophic markers
- Reversion of multiple markers at same time now much lower
- Genetic analysis possible by looking at multiple markers
How to perform an E. coli genetic cross?
- Take two strains, each with two different nutritional requirements
- Grow together overnight in complete medium
- As controls, grow each strain seperately - Collect Cells by centrifugation
- Plate on minimal medium
- Colony formation indicates recombination
- Controls for reversion are colony formation from each strain alone - Obstain approx. 1 colony/10^7 bacteria plated
U-tube experiment
- Filter blocked direct bacterial contact
- push medium back and forth between chambers separated by a filter
- DNA molecules could pass between chambers, but **NO actvity **
Identification of F, the fertility factor in male (donor) strains
- Hayes and the Lederbergs indepedently accomplished experiments leading to identification of fertility factor, F
- F factor must be present in one parent in the corss
- Cross must be F+ x F-
- F factor is transmitted by contact
F factor
- F+ x F-
- Progeny change rarely with respect to auxotrophy, but are frequently F+
F factor conjugation
What is Pilus?
- Pilus establishes contact between F+ and F= cells
- After contact is made, pilus retracts, bringing cells into close contact
- F+ cell assembles Type IV secretion apparatus
- F factor replicates by rolling circle mechanism and the “displaced” strand is transferred to the F= recipient
- Strand is used as template to produce double-strand DNA recipient
How was F factor isolated?
1961
- S. Falkow and L.S. Baron mixed F+ *E. coli *with *Serratia marcesscens *
- F+ was transferred into this distantly related species
- Base composition differences enabled purifiaction in CsCl gradients
- Measurement of the amound of DNA in the satellite band led to estimate of size of 60 kb
Conjugation between F+ cell (donor) and F- cell (recipient)
What are the 5 steps
- The pilus retracts
- Cell pairs are stabilized. F plasmid nicked in one strand
- Transder of one strand from F+ cell to F- cell. F plasmid simultaneously replicated in F+ cell
- Synthesis of the complementary strand begins in the recipient cell
- Completion of DNA transfer and synthesis. Cells separate.
What are the majority of genes in the region of F factor with genes needed for conjugation?
There are trb and tra, the majority is tra
traA: Encodes pilin protein
traK, traB, traP: Encode proteins that are components of the type IV recretion system
traD: Encodes coupling protein
tral: Encodes relaxase
F+ x F- mating
- A copy of the F factor is transferred to the recipient and and does not integrate into the host chromosme
- Donor genes usually not rasnferred
Steps of the transfer of F- recipient
Start with Hfr cell (donor) and F- cell (recipient)
1. The F plasmid is nicked in one strand
2. F is transferred, followed by chromosomal DNA
3. Synthesis of second strand in recipient and donor cells
Hfr conjugation - defined
- High frequency of recombinates = Hfr
- Donor chromosomal genes are transferred with great efficiency but F- recipient is not converted to F+
- Donor is Hfr strain; mating event is called Hfr conjugation
- Hfr strains contain the F factor integrated into their chromosome
Hfr conjugation steps
How chromosome is trasnferred to F- cell
tra (trasnfer function) of F factor still function from chromsomal location
Chromosome is strasnferred to F- cell
1. Direction of transfer depends on the orientation the integrated F factor
2. Transfer of the chromsome takes apporximately 100 min
3. “interrupted” mating enables mapping
4. F factor genes are the alst to be trasnderred, which is why F- recipient is only rarely converted to F+
Transduction via lysogenic phage
Transduction is a common mode of horizontal gene transfer
- Transduction is mediated by viruses (phage)
Phage can have lysogenic stage as well as a lytic cycle
Such phage do not immediately begin their lytic cycle after entering the host
- Phage that do not have to immediately undergo lytic cycle are called “temperate” phage
Prophage
“Lysogenic” phage can insert their genome into the bacterial genome
* This form of phage is called prophage
* The phage genome passively replicates with the host genome
* Under specific inducing conditions, the prophage excise and begin their lytic cycle
Who and how was generalized transduction discovered?
- Joshua Lederberg and Norton Zinger were attempting to analyze conjugation in Salmonella typhimurium
- Duplicating the procedures used to show conjugation in* E. coli*
- Mix two strains that were each multiply auxotrophic
- Recover prototrophs at high frequency
- Original conclusion was that this was evidence for conjugation: wrong
How is the U-tube experiment used to produce Salmonella prototrophs?
physical proimity is not needed
- Filter blcoked direct bacterial contact
- Nevertheless, Salmonella prototrophs were produced at high efficiency
- Phage P22 is small and passes through the filter
- P22 prophage in parental chromosomal DNA provides souce of phage
Transduction*
- Transfer of bacterial genes by viruses (phage)
- Bacterial genes are inforporated into the phage genome by “errors” that occur as the phage propagates
- The phage that carry the bacterial DNA transder that DNA to a new bacterium upon infection
Generalized transduction
How this occurs
- Occurs during lytic cycle of virlent phages and lytic cycle of some temperate phages
- Host genome is broken into pieces during lytic phage cycle
- The phage particles can inject the bacterial DNA into another bacterial cell
- The transduced DNA from the donor genome must be incorporated into the recipient chromosome to become stably transferred
- If the DNA is not stably transferred, the recipient is an “abortive transductant”
- Molecular biologists expoit transucing phage particles as “delivery vehicles” to clone DNA
Phage Particles
- Some peices from host genome breaking into peices during lytic phage cycle are accidentally packed into phage particles
- There is no phage genome in these generalized transduction phage particles
Whe use genralized transduction mapping?
- Used to establish gene linkage
- Expressed as frequency of cotransduction
What happens if two genes are close togher for generalized transduction mapping?
There is an increased likelihood that they will be carried on a single DNA fragment in a single transducing particle
* A P22 particle can transduce approx 1% of the Salmonella genome (1% is a lot, that 40-50bp)
* Phage P1 can transduce approximate 2-2.5% of the *E. coli *genome
Specialized Transduction
- Occurs only with temperate phages that have established lysogeny
- Only specific portion of bacterial genome is transderred
- That part of the genome adjacent to the integration site of the prophage
- Occurs when prophage is incorrectly excised
What did Antony van Leeuwenhoek invent and discover with his invention?
Microscope
* Dutch draper
Discovered:
* Bacteria
* Sperm cells
* Blood cells
* Protists
Chromalveolata Supergroup
Alveolata
Consists of:
Consists of:
* Dinoflagellata
* Ciliophora (ciliates)
* Apicomlexa
Cilia Structure
- 9 outer tubules
-1 compelte, 1 incomplete - 2 complete core tubules
- Dynein arms
What’s the direction of cilia’s dynein?
Dynein walks in the (-) minus direction along the microtubule
Trichocysts
organelle that releases long filamentous proteins. Trichocysts are released upon mechanical or chemical stimulation (self defence)
Ameobozas supergroup
Eumycotozoa group
Consists of:
Consists of:
* Myxogastria
* Dictyostelia
What are dictyostelium discoideum?
- “smile molds”
- a model orgamism for reserch
- Feed on bacteria and yeast
- Exhibits unicellular and multicellular behavior
- The change from unicellular to mulitcellular is triggered on nutreint limitation
Life cycle of a Dictyostelium
6 stages
- Free-living amoeba is released
- Ameobas begin to congregate
(Amoeba mass forms) - Moving amoeba mass is called a slug
- Slug begins to right itself
- SLug is stransformed into spore-forming body, the sorocarp
- spores
cAMP
- Cyclic adenosine monophosphate
- Signaling molecule in MANY organisms
Stalk and spore development
youtube vido on slides
- Fruiting body formation
- Spores suspended in a slime droplet
Archaeplastida Supergroup
Chloroplastida
- “green algae”
- Grows in fresh/salt water, soil and in/on other organisms
RuBisCO
- Ribulose-1,5-bisphosphate carboxylase oxygenase
- Considered the msot abundant protein on Earth
- Catalyzes the addition of CO2 to ribulose 1,5 bisphosphate (CO2 fixation)
Fungi
- Found where moisture, the appropriate temp and suitable organic nutrients are present
- Molds and yeasts
- Molds exist primarily as filamentous hyphae
- mycelium=a mass of hyphae
- Fungi secrete exoenzymes to digest insuluble matter, then absorb the solubilized nutrients
- Often display two modes od spore formation for reproduction
- Asexual (genetative)
- Sexual
Fungus (pl., fungi)
- Eukaryotic
- Spore-bearing
- Chemoorganoheterotrophs with absorptive metabolism
- Lack chlorophyll
- Repro sexually and asexually
- Belong to kingdom* Fungi (Eumycota)* within domain Eukarya
Mycology
Study of fungi
Mycotoxicology
Study of fungal toxins (myucotoxins) and their effects
Mycoses
DIsease caused by fungi
Distribution of fungi
- Primarily terrestrial: few equatic
- Many are saprophytes
- Some are pathogenic in plants or animals
- some form associations: Mycorrhizae, Lichens
saprophytes
decompose dead oganisms
Mycorrhizae
associations with plant roots
Lichens
associations with algae or cyanobacteria
Importance of Fungi
* Beneficial impact
* Research
* Detrimental impact
Beneficial impact
- decomposers
- industrial fermentations
* fermented foods and drinks
* steroids, antibiotics, other drugs
Research: Model eukaryotic organisms
Detrimental impact
- Major cause of plant diseases
- Cause of many animal, including human, diseases
Yeast
- Unicellular fungi
- Reproduce asexuallu, often by budding
- Reproduce sexually by formation of spores
Filamentous fungi - Molds
Hyphae
* filaments of mold
* coenocytic - they contain multiple nuclei in the same “cellular compartment”
* Septae may be present to help isplate the compartments in case of rupture
Mycelium
* Bundles or tangled masses of hyphae
What’s Dimorphism in fungi, animals, and plants?
- Some fungi change from yeast form (Y) to mold (filamentous) form
- In animals, typically yeast in host and filamentous outside host
- In plants, filamentous in host and yeast outside host
Nutrition and Metabolism of fungi
Most are saprophytes
- Obtain nutrients fromd ead organic material
- Secrete hydrolytic enzymes
Chemoorganoheterotrophs
Most are aerobic
- some are facltative anaerobes
- obligate anaerobic fungi are found in rumen of cattle
who performs asexual reproduction?
- Yeast: division by fussion or budding
- Filamentous fungi: extension of hyphae
- Asexual spore production
Sexual reproduction
Involves union of compatible nuclei followed by formation of sexual spores (zygospores, asconspores, or basidiospores)
Uredinimycetes and Ustilaginomycetes
- Plant pathogens causing “rust” and “smuts”
- Some Urediniomycetes are also human pathogens
Lambda phage
Genome
* ~48,000 bp
* Package as linear DNA, replicated as circular
Why is the 12 bp sequence C:G rich?
GC is a stronger bond with 3 hydrogen bonds, hydrodize much stronger
Virulence vs. Lysogeny
- Virulence: replication, synthesis, assembly & lysis
- Lysogeny: For lambda, DNA is inserted into att sites on teh bacterial chromosome. DNA is then replicated as the bacteria replicates
what dictates lysogeny
cl and Cro
- 2 proteins that influence viral lifecycle
If cl> Cro
lysogeny
If Cro > cl
lytic
cll
- protects from degradation
- activates expression of cl and genes required for integration
cl and Cro
- cl represses Cro
- Cro activates the expression of genes required for replication, assembly and lysis and represses cl
Clustered, Refularly Interspeaced Short Palindromic Repeasts (CRISPRs)
- Short RNA-based defense systems in bacteria and archaea
- Provide immunity against viruses, plasmids or other mobile genetic elements
- 3 phases: Adaptation, crRNA Biogenesis, Invader Silencing
Features of CRISPR loci
- Leader AT-rich, not conserved
- Repeat - 23-50 nt, number of repeats is variable
- Spacer - share sequence identity to fragments of plasmids or phages
- cas genes - CRISPR associated genes
Phage titering
what is it? the porcess of plating it?
- The number of pahges in a given solution is determined using “plaque forming units” (PFU)
- The cell-phage mixture is poured onto a soldified nutreint agar plate.
(Mixture containing molten top agar, bacterial cells, and diluted phage suspension) - The mixture is left to solidify
(sandwich of top agar and nutreint agar on bottem) - Incubation allows for bacterial growth and phage replication
(phage plaques surrounded by lawn of host cells)
Phage therapy
- Phage therapy is the use of bacteriophage to treat infections
- First used in 1919 France to treat a boy with severe dysentery who recovered from the infection
Bacteriophages vs. Antibiotics
Advantages of bacteriophages
- Very specific (affect only targeted bacterial species)
- Reolicate at the site of infection
- Occur naturally (easy go locate)
- Safe (no reports of serious adverse effects)
- Active agaisnt antibiotic-resistance bacteria
Bacteriophages vs. Antibiotics
Disadvantages of bacteriophages
- Additional reserch required (lack of studies)
- Development of phage resistance and phage-neutralizing antibodies
- not accessible to intracellular pathogens
- Difficult to administer (special traning required)
- Can transfer toxin genes between bacteria
Viral Genome
Different types of DNA
- Double stranded (dsDNA -> herpesviridaw, poxviridae)
- Single stranded (ssDNA -> Phage M13)
Viral Genome
Types of RNA
- Double stranded (dsRNA -> reoviridae (HIV))
- (+) stranded (retroviridae, picornaviridae… coronavirus)
- (-) stranded (filoviridae, rhabdovirdae)
Viral lifecycle
- Attachment (absorption)
- ENtry
- Synthesis
- Assembly
- Release
What are the entry point techniques?
- fusion
- endocytosis (enveloped and non-enveloped)
Entry by fusion
- virus’s envelope spikes bind to receptors on surface of host cell
- Lipid bilayer of viral envelope** fuses** with host cell membrane
- Nucelocapsid is released into the cytoplasm
Entry by endocytosis (enveloped virus)
- Virus’s envelope spikes bind to receptors enriched inthe membrane of a coated pit on the cell’s surface
- Binding to the recepor triggers receport mediated endocytosis
(endosome intermediate) - increased acidity allows nucleocapsid to escape from the endosome and entrer the cytoplasm
Entry by endocytosis (non-enveloped)
- Virus’s capsid proteins bind to recepors on cell surface and triggers receptor0mediated endocytosis
- Nucleic acid is extruded from the endosome into the cytoplasm
Release- enveloped viruses
Many enveloped viruses are released from infected cells by maturing and budding at the plasma membrane. During this process, viral core components are incorporated into membrane vesicles that contain viral transmembrane proteins, termed ‘spike’ proteins.
steal some of host cell membrane
Get a free infectious virion eith envelope
Release- nonenveloped viruses
cell lysis
daDNA virus
- Herpes Simplex Virus (HSV)
- HSV-1 : mainly cold sores
- HSV-2: mainly genital herpes
Herpes infection symptoms
- Gingivostomatisis (cold sore)
- Herpetic keratitis (eye)
- Genital Herpes
What are the componets of the viral strucutre?
- Envelope proteins (gB-gN)
- Lipid evelope
- Tegument
- DNA
- Nucleocapsid
HSV Entry
- Once in the cell, the viral capsid traffics to the nucleus
- Uses microtubules to get there
- Once outside the nucleus, the DNA is released and it is inserted into the cell’s nucleus
HSV transcription
3 stages
1. Immediate-early
2. early
3. late
HSV Replication
- Once HSV replicates to a few copies/cell the viral DNA is then replicated by rolling circle replication
- The linear DNA is cut, packaged, and then the virus buds from the nucleus
