Horizontal Gene Transfer and CRISPR/Cas Flashcards
Mutation
Heritable change in DNA sequence
Alleles
Alternate forms of genes
Mutation can give rise to new ____.
Phenotypes
Vertical gene transfer: what is it, what domain of life can use it
Sexual reproduction: new combinations of genes arise when gates from parents fuse
Eukarya
How do bacteria and archaea generate genetic variability without sexual reproduction?
Horizontal gene transfer
Horizontal gene transfer: how it works
Genes are transferred from one independent organism to another
3 mechanisms of horizontal gene transfer
Conjugation
Transduction
Transformation
Conjugation
DNA transfer through direct cell-cell contact
What 2 things does conjugation require?
Pili
Plasmids
Conjugation is the major mode of spreading what kind of genes?
Antibiotic resistance
Plasmids: single or double stranded DNA? Linear or circular?
Double stranded, circular
Do plasmids rely on chromosomes, or are they separate?
Separate/ extrachromosomal
Plasmids carry genes that confer _____.
Advantage
Plasmids can be transferred by ______.
Conjugation
Plasmids are replicons. What does that mean?
They have their own ori
Episomes
Plasmids that exist with or without integrating into chromosome
Well-known example of conjugative plasmid: name and species that has it
F (fertility) factor
E. coli
Conjugation can also be referred to as _____.
Mating
Step 1 of conjugation
Pilus extension from donor (F+) to recipient (F-)
Step 2 of conjugation
Pilus retracts
F factor begins replication and transfer
Step 3 of conjugation
Plasmid-encoded endonuclease nicks one strand of F factor
F factor is replicated and transferred
Single strand enters recipient
Step 4 of conjugation
New complementary strand is made through rolling circle replication in both donor and recipient
Hfr cell: what does Hfr stand for?
High frequency of recombination
An Hfr cell does what with the F factor plasmid? What does this mean for gene transfer?
Integrates it into chromosome
Integrated F factor can also be excised out of chromosome, taking part of chromosome with it
Example of bacteria that can conjugate with plant cells
Argobacterium tumefaciens
Argobacterium tumefaciens causes what disease in plants?
Crown gall disease
What type of plasmid does Argobacterium tumefaciens have?
Tumor-inducing plasmid (Ti)
How does Argobacterium tumefaciens’s Ti plasmid work (3 steps)?
Piece of Ti called T DNA is transferred by conjugation from bacterium into plant cell
T DNA integrates into plant genome
Ti genes are expressed, causing tumor growth
What does the 2-component system of Argobacterium tumefaciens do?
Senses compounds released by wounded plant, then activates transcription of transfer genes
T DNA genes of Argobacterium tumefaciens perform what 2 functions?
Direct plant cells to overproduce phytohormones (uncontrolled growth leads to tumor formation)
Stimulate plant cells to make opines (carbon and nitrogen source for bacteria)
If T DNA was replaced with drought resistance gene, what would happen to plant? This mechanism can be used for____ ______ of plants.
Plant would become drought resistant
Genetic engineering
Transformation
Uptake of free DNA from environment
Competent cell
Can naturally take up DNA from environment
What 4 bacteria genera are known to be naturally competent?
Gram positive: Streptococcus, Bacillus
Gram negative: Haemophilus, Neisseria
Artificial transformation
Making bacteria competent that aren’t naturally competent
2 techniques of artificial transformation
- Calcium chloride: makes cells more permeable
2. Electroporation: pulsing high voltage that creates temporary holes in cell wall and plasma membrane
Artificial transformation is a critical step in what?
Cloning
2 possible outcomes of DNA transformation
- Stable transformation: DNA is integrated into chromosome by homologous recombination
- Unsuccessful transformation: DNA isn’t integrated into chromosome and is instead degraded
Protein that integrates DNA by homologous recombination in transformation
RecA
What bring DNA into the cell during transformation?
Membrane-bound protein complexes
How is DNA changed when being brought into the cell during transformation? What is the significance of this change?
Nuclease converts double stranded DNA into single stranded DNA
Single stranded DNA is easier to incorporate into genome
Bacteriophages
Viruses that infect bacteria
Bacteriophages are _____ and _____.
Abundant
Diverse
How many bacteriophages per liter of sea water?
10 billion
Bacteriophages impact _____ and _____ of microbial communities
Composition
Behavior
Transduction
Bacterial gene transfer by phages
2 major phage types
Virulent
Temperate
Virulent phages reproduce through what type of cycle?
Lytic (causes bacterial cell lysis)
Temperate phages reproduce through what type of cycle?
Lysogenic
Steps of lytic cycle
- Attachment of phage to host cell receptor
- Phage injects DNA into cytoplasm
- Phage DNA directs synthesis of many new phages; degradation of bacterial genome
- Cell lyses and releases new phages
Steps of lysogenic cycle
- Attachment of phage
- Phage injects DNA into cytoplasm
- Prophage is created: phage genome is integrated into bacterial chromosome
- Prophage DNA is copies when cell divides
What triggers excision of phage DNA from host genome in lysogenic cycle?
Environmental stressors such as UV light
Generalized transduction occurs during which cycle?
Lytic
Generalized transduction: how does it work?
During viral assembly, pieces of degraded host DNA are mistakenly packaged into phage
During generalized transduction, is just bacterial DNA packaged into phage, or can it be a mixture of viral and bacterial DNA? Can any part of bacterial genome be transferred?
Can be just bacterial DNA or mixture of viral and bacterial DNA packaged into phage
Any part of bacterial genome can be transferred
Specialized transduction occurs during which cycle?
Lysogenic
Specialized transduction: how does it work?
Prophage incorrectly excises, taking part of bacterial genome with it
3 defense strategies of bacteria against bacteriophages
- Surface alterations (prevent phage attachment)
- Restriction-modification systems (modify bacterial genome, then use restriction enzymes to degrade unmodified foreign DNA)
- CRISPR/Cas systems
CRISPR/Cas system acts as prokaryotic _____ system.
Immune
CRISPR/Cas system is DNA or RNA based?
RNA
CRISPR: what does it stand for?
Clustered regular interspaced short palindromic repeats
CRISPR: what is it?
Clusters of short DNA sequences found in genomes of many bacteria and archaea
Upon phage infection, bacteria do what with sequences of viral DNA?
Capture and integrate them into their own genetic material, placing them in between the CRISPR repeats
Next time bacteria encounter the phage, what do they do?
Transcribe DNA in CRISPR clusters, making RNAs complementary to infecting viral DNA sequences
Complementary RNAs synthesized from CRISPR clusters guide what kind of proteins to viral DNA? What do those proteins do to the DNA?
Cas proteins
Cut invading DNA
How do Cas proteins enable integration of phage DNA into CRISPR locus?
Cleave phage DNA into bits
6 steps of CRISPR/Cas operation
- Phage injects its DNA into bacterial cell
- DNA is cleaved and integrated into CRISPR locus as spacers in between repeats
- CRISPR locus is transcribed into pre-crRNA
- Pre-crRNA is cleaved into many transcripts, each containing a different phage’s genetic material
- Cas protein takes up crRNA, which guides Cas to phage
- Cas cuts phage’s DNA
6 steps of CRISPR/Cas-mediated gene editing
- Make synthetic guide RNA that matches DNA to be cut from genome
- Add Cas nuclease
- Deliver both guide RNA and Cas into cells
- Guide RNA directs Cas to genomic sequence to be cleaved
- Cas cuts DNA at desired site that is homologous to guide RNA
- Donor DNA is used to repair cut
What is an alternate method of injecting separate guide RNA and Cas into cells?
Injecting a plasmid that encodes both Cas and guide RNA into cells