Chapter 9 - Bacterial and Viral Genetic Systems Flashcards
What are some examples of important contributions bacteria and viruses have made to genetics?
- Evidence for nature of genetic material (DNA)
- First characteristics/definitions of gene
- First evidence for gene interactions at molecular level
- Basic principles of inheritance
What makes bacteria valuable research objects?
- Small size
- Rapid reproduction
- Selective media that can easily identify presence of an active allele
- Simple structures/physiology
- Genetic variability
What are bacterial viruses/bacteriophages?
- Reproduce by infecting bacterial cells
- Helped discover some important genetic concepts
How do bacteria grow in liquid media?
- Inoculate medium with bacteria
- Bacteria grow and divide
How do bacteria grow in solid media?
- Growth medium is suspended in gelatin-like agar
- Add a dilute solution of bacteria to petri plate
- Spread evenly with glass rod
- After incubation for 1-2 days, bacteria multiply, forming visible colonies
What do bacteriophages produce on plates with dense bacterial cultures?
- Clearances/plaques within hours of infection
What does Bacteriophage T4 consist of?
- Protein head
- 168,800 base pairs and 150 characterized genes
- Fairly large/complex genome for a virus
- Phage goes through lytic phase (lyses cell to infect other cells); quick/simple experiments
- No dormant phase
- Tail fibres land and hook onto plasma membrane
What does Bacteriophage Lambda consist of?
- 48,502 base pairs and 50 genes (about 1/3 of T4)
- May be lytic or lysogenic (inserts DNA into host and goes into latency/dormancy)
How does a retrovirus work?
- Attaches at receptors in membrane
- Viral core enters host
- Viral RNA uses reverse transcriptase to make complementary DNA
- Reverse transcriptase synthesizes 2nd DNA strand
- Viral DNA enters nucleus and is integrated into host chromosome (forming provirus)
- Proviral DNA is transcribed into viral RNA on activation
- RNA is translated in cytoplasm
- Viral RNA, proteins, new capsids and envelopes are assembled
- Assembled virus buds from cell membrane
What is the lytic cycle?
- Phage binds to bacterium
- Phage DNA enters host
- Host DNA is digested
- Phage DNA replicates
- Host cell transcribes/translates phage DNA -> proteins, phages are assembled
- Phage encoded enzyme causes cell to lyse
- New phages released to start cycle again
What is the lysogenic cycle?
- Phage binds to bacterium
- Phage DNA enters host
- Phage DNA integrates into bacterial chromosome (becomes prophage)
- Prophage is replicated as part of bacterial chromosome (can be many cell divisions)
- Prophage may separate from chromosome and cell will enter lytic cycle after dormancy
Why would an infected cell go through dormancy?
- Nutrients are scarce
- Host is sick
- Not happy with conditions
What do bacterial genomes look like?
- Circular molecules
- Several million base pairs
- Double-stranded DNA molecule
Where is additional genetic material found in bacteria?
- Plasmids (small circular DNA)
- Can replicate independently of bacterial chromosome
What are episomes?
- Subclass of plasmid
- Large circular DNA
- Can integrate into bacterial chromosome for replication or remain separate
Is bacterial recombination possible?
- Yes, but not through meiosis/mitosis
Is gene transfer unidirectional or bidirectional?
- Unidirectional
Gene mutations can be readily observed via… (4)
- Colony colour and morphology
- Nutritional mutants (cannot metabolise certain sugars)
- Prototrophs (can make all AAs) and auxotrophs (cannot make certain AAs, need to be added to medium)
- Antibiotic resistance
What happens when a bacterial culture is plated onto petri dishes containing and lacking leucine? Why is this useful?
LACKING
- Only leu+ grow
CONTAINING
- leu+ and leu- grow
- Leucine auxotrophs (leu-) are recovered and cultured for further study
- A colony that grows only on supplemented medium has a mutation in gene that encodes synthesis of essential nutrient
What are the 3 parasexual processes through which bacteria exchange genetic material?
- Conjugation
- Transformation
- Transduction
What happens in conjugation?
- Direct transfer of DNA from one cell to another via cytoplasmic bridge
- ‘sexual’ reproduction mediated by Fertility/F factor (an episome)
- Cytoplasmic bridge forms b/n bacteria cells
- DNA replicates and transfers from one cell to another
- Transferred DNA replicates
- A crossover in the recipient cell leads to the creation of a recombinant chromosome
- Cell contact IS required
- It is NOT sensitive to DNase
What happens in transformation?
- Transfer of free piece of DNA from one bacterium into another
- Competent bacterial cells take up DNA from environment
- Transferred DNA can be stably inherited
- Naked DNA is taken up by recipient cell
- Crossover in bacterium leads to recombinant chromosome
- Cell contact is NOT required
- It IS sensitive to DNase
What happens in transduction?
- Transfer of genes from one cell to another via bacteriophage
- Bacteriophage can ‘hijack’ bacterial chromosome genes during phage assembly and donate hijacked genes to another cell
- Virus attaches to bacterial cell, injects its DNA and replicates (taking up bacterial DNA)
- Cell lyses
- Virus infects new bacterium carrying bacterial DNA with it
- Crossover in recipient leads to recombinant chromosome
- Cell contact is NOT required
- It is NOT sensitive to DNase
- Transfer of genes from donor strain and recombination produce transductants in recipient bacteria
How do we establish the need for contact?
- U-tube experiment
- 2 auxotrophic strains were separated by filter
- No prototrophic bacteria were produced
- Therefore genetic exchange requires direct contact b/n bacterial cells
What is a competent bacterium?
- Can bind exogenous DNA and transport it into the cell
What can transformation be used for?
- To determine distance b/n genes
- Only 0.2-0.5% of chromosome undergoes transformation
- If 2 genes are close, they will be present on same piece of DNA
- Transformation is measured as a change in phenotype and it occurs with a certain frequency
How is the rate of cotransformation related to distance between genes?
Rate of cotransformation is inversely proportional to distance between genes
- As distance increases, cotransformation decreases
How can an F-factor exist?
- As extra-chromosomal DNA
OR - Insert into the bacterial chromosome and replicate as part of it
What is a F- cell?
No F factor
What is a F+ cell?
Autonomous F factor
What is a Hfr cell?
Integrated F factor
What is conjugation in E. coli mediated by?
Cytoplasmic ‘conjugation’ channel
What happens in a F+ x F- cross?
- F pili of F+ make contact with F- to pull cells together
- Genes on F factor direct synthesis of conjugation bridge
- One strand of DNA is cleaved at origin of F factor
- Rolling-circle replication transfers one strand into F-
- Replication occurs in both cells during transfer
- Ends with 2 F+ bacteria
What happens in a Hfr x F- cross?
- F is nicked and 5’ end moves into F- cell
- Transferred strand replicates and crossing over takes place b/n donated Hfr and original F- chromosome
- Crossing over may lead to recombination
- Linear chromosome is degraded
What happens in Hfr1?
- F is integrated b/n leu gene and azi gene
- So the genes are transferred beginning with leu
What happens in Hfr5?
- F is integrated b/n thi and his
- F has the opposite orientation in this chromosome, so the genes are transferred beginning with thi
Conjugation b/n an F+ and F- cell usually results in what?
2 F+ cells
What is a plasmid?
- An extra-chromosomal circular piece of DNA
- Can replicate independently of chromosome
- In general, not required for survival of host cell and serve as auxiliary genetic elements
What is an episome?
- Genetic element that is not essential to the host and that can either replicate autonomously or be integrated into the bacterial chromosome
What are examples of plasmids and episomes in E. coli?
- F factor (episome; fertility factor)
- R plasmids (resistance plasmids)
How are distance between genes and cotransduction related?
- Genes close to one another are more likely to be cotransduced
- Rate of cotransduction is inversely proportional to distance b/n genes
