Bacterial and Viral Genetic Systems

Question 1

What are bacteria and viruses in genetics?

Answer 1

-have made most important contributions to the science of genetics
-evidence for the nature of genetic material (DNA)
-first characteristics and definitions of a gene
-first evidence for gene interactions at the molecular level
-basic principles of inheritance

Question 2

What makes bacteria valuable research objects?

Answer 2

-small size can house 1 billion in a small test tube does not take up space
-rapid reproduction (large sample size, life cycle of mins)
-selective media that can easily identify the presence of an active allele
-simple structures and physiology
-genetic variability

Question 3

What are bacterial viruses (bacteriophages)?

Answer 3

-bacteriophages reproduce by infecting bacterial cells
-several important genetic concepts have been discovered through studies of bacteriophages

Question 4

How do bacteria grow in liquid or solid media?

Answer 4

-sterile liquid medium inoculate medium with bacteria
-bacteria grow and divide
-growth medium is suspended in gelatin-like agar
-add a dilute solution of bacteria to petri plate
-spread bacterial solution evenly with glass rod
-after incubation for 1-2 days bacteria multiply forming visible colonies
-dense plate: every colony is where a single bacterium dropped down onto agar
-left overnight
-need a sterile bacteria
-clear medium is opaque versatile and can grow on petri plate
-add powder to liquid so agar powder dissolved but a room temp

Question 5

How do we study viruses?

Answer 5

-must produce plaques
-circles are clearances where a virus infected one bacteria and it killed everything
-when you add liquid with bacteriophage
-produce clearances (plaques) on plates with dense bacterial cultures within hours of infection

Question 6

What is the formation of a bacteriophage?

Answer 6

-head contains the genome
-hydrophobic tail
-human genome has 3 billion base pairs and bacteria has ten times less
-phage goes through lytic phase meaning it lyses the cell to infect other cells

Question 7

What happens in a eukaryotic cell RNA virus?

Answer 7

-1. Virus attaches to host cell at receptors in the membrane
-2. Viral core enter the host cell
-viral RNA uses reverse transcriptase to make complementary DNA and viral RNA degrades
-reverse transcribing th eRNA genome into DNA
-viral DNA enters the nucleus and foms a provirus
-activation proviral NA is transcribed into viral RNA exported to cytoplasm
-viral RNA is translated
-viral RNA proteins, new capsids and envelopes are assembled
-virus buds from the cell membranes
cells want to preserve DNA and limit the amount of mutations
-require more mutations therefor adapt more quickly to host genome

Question 8

What is the lytic cycle?

Answer 8

-phage encoded enzyme causes the cell to lyse 5
-host cell transcribes and translates the phage DNA producing phage proteins 4
-phage DNA replicates 3
-host DNA is digested 2

Question 9

What is the lysogenic cycle (dormant)?

Answer 9

-phage DNA integrates into the bacterial chromosome and becomes a prophage
-prophage is replicated as part of the bacterial chromosome
-virus does not want to stay here forever
-prophage may separate from the chromosome and the cell will enter the lytic cycle (can choose to enter)
-if host is declining ATP levels are low and shows that it is likely to go to the dormant phase
Bacteriophage lambda (prophage lytic genes are suppressed) can enter lytic like T4 or can enter lysogentic pathway

Question 10

What are genetics of Bacteria?

Answer 10

Genomes are circular molecules of several million base pairs called bacterial chromosomes
-additional genetic material resides in plasmids (small circular DNA) can give you an advantage
Episomes (a subclass of plasmid) are large circular DNA that can integrate into the bacterial chromosomes for replication or remain separate
- bacteria do not have meiosis hence they cannot have chiasmata
-bacterial recombination is possibl ethrough processes
Gene transfer in bacteira is unidirectional, always a donor and recipient, never a trade

Question 11

What are the phenotypes in bacteria?

Answer 11

-gene mutation can be readily observed via:
-colony colour and morphology
-nutritional mutants can not metabolise certain sugars like lactose or galactose
- prototrophs -bacterial strain that is self-sufficient and can synthesize all essential nutrients
-auxotrophs mutant that can not synthesize amino acids
-antibiotic resistance
-a colony that grows only on the supplemented medium has a mutation in a gene thtat encodes the synthesis of an essential nutrient

Question 12

What are the parasexual processes that bacteria exchange genetic material can go through?

Answer 12

1. Transformation
2. Conjugation
3. Transduction

Question 13

What is transformation?

Answer 13

-transfer of a free (out of cell) piece of DNA from one bacterium into another
-have on copy and allelel in every gene
-naked DNA is taken up by the reciepeint ell
-crossover in the bacterium leads to the creation of a recombinant chromosome
-DNA environment is chopped up in tiny pieces
-picks up a free allele and can lead to Z copies
-no cell contact is required and it is sensitive to Dnase controlled enzyme that chops up the DNA
-it is free and loose to the environment

Question 14

What is conjugation?

Answer 14

-direct transfer to DNA from one cell to another via the establishment of a cytoplasmic bridge
-donor cell (bacterial chromosome) and recipient cell
-cytoplasmic bridge forms
-DNA replicates and transfers from one cell to the other
-donor gives copy does not lose allele
-transferred DNA replicates where recombination can occur
-Cross-over in the recipient cell leads to the creation of a recombinant chromosome
-degraded DNA ways bacteria can gain new alleles
-cell contact is required and it is not sensitive to the DNase* (controlled enzyme that chops up the DNA)

Question 15

What is transduction?

Answer 15

-transfer of genes from one cell to another via bacteriophage (vector)
-a virus attaches to a bacterial cell injects DNA and replicates taking up the bacterial DNA
-the bacterial cell lyses
-the virus infects a new bacterium carrying bacterial DNA with it
-a crossover in the recipient cell leads to the creation of a recombinant chromosome
-DNA is never free in the environment no cell contact is required*
-it is not sensitive to DNase*

Question 16

How do we establish the need for contact: The U-tube experiment?

Answer 16

Methods are auxotrophic strain A, which has leucine + and can synthesize
-auxotrophic strain B, which never genetically change (bacteria and has airflow)
The added virus gets both leucine and glycine so transduction is possible
-two auxotrophic strains were separated by a filter that allowed mixing of medium but not bacteria
No prototropic bacteria were produced
-prevents conjucation and separates strands; pores are too small for cells to pass
-free DNA can travel through the barrier
-leu is negative and defects the pathway glycine is positive
-genetic exchange requires direct contact between bacterial cells

Question 17

What is more of an explanation of transformation?

Answer 17

-bacteria can pick up free DNA but only happens when it needs too
-a process by which “ competent” bacterial cells take up DNA from the environment
-transferred DNA can be incorporated into the recipient bacterial chromosome
-cell only prepares for bacteria if they think it will be beneficial (competent)
-only one of the two sister chromatids gets the new allele
-one strand of the DNA fragment enters the cell the other is hydroylzed
-single-stranded fragment pairs with the bacterial chromosome and recombination takes place
-the remainder of the single-stranded DNA fragment is degraded
-one cell is transformed one if not

Question 17

What can be used to determine the distance between bacterial genes?

Answer 17

-form of chromosome mapping
-only about 0.2-0.5 % of the entire bacterial chromosome undergoes transformation
-if two genes are very close they will present on the same piece of transforming DNA
-measured as a change in phenotype and occurs with a certain frequency

Question 18

What is the rate of cotransformation?

Answer 18

-DNA from a donor cell is fragmented wild type for genes A B & C
-recipient cell has recessive version of donor alleles
-donor cell has different sized pieces
-fragments are taken up by the recipient cell
-after entering the cell the donor DNA becomes incorporated into the bacterial chromosome through crossing over
-genes are close to one another on the chromosomes are more likely to be present on the same DNA fragment and be recombined together
-rate of cotransformation is inversely proportional to the distances between genes

Question 19

What is conjugation?

Answer 19

-sexual reproduction mediated by the fertility of F factor (this is an episome) plasmids that can be incorporated into genome
-an F factor can exist as an extra-chromosomal DNA or it can insert into the bacterial chromosome and replicate as part of it
F- cell chromosome
-F+ cell donor free DNA the autonomous F factor enters cell
-Hfr cell (high frequency) intergrated into chromosome

Question 20

How do mating and conjugation in F+ x F- crosses occur?

Answer 20

-F+ donor cell and F- recipient chromosome
-the F pili of the F+ donor cell makes contact with the F- recipient cell and pulls the cells together
-genes of the F factor direct the synthesis of the conjugation bridge
-one strand of DNA is cleaved at the origin of replication of the F factor
2. Conjugation bridge provides channel between cells
-rolling-circle replication transfers one strand of the F factor
-replication of the F factor occurs in both cells
-one strand is synthesized in each cell during transfer
-the F factor into the recipient cell replication of the F factor occurs during transfer
-tranfer of the F factor is complete yielding two F+ bacteria
-copies it is not losing it is just replicated strand both have F+ cells

Question 21

How does the process of conjugation occur?

Answer 21

-F is nicked and the 5’ end moves into the F- cell
-the transferred strand replicates and crossing over takes place between the donates Hfr chromosome and the original chromosome of the F- cell
-when conjugation stops you get a Hfr chromosome
-crossing over may lead to the recombination of alleles
-incomplete copy of the fertility factor
-the linear chromosome is degraded
-only benefit is that you get a new chromosome
-*conjugation is short in time because the channel is very fragile and can break easily

Question 22

How does Hfr1 and Hfr5 occur?

Answer 22

-transfer always begins within F and the orientation of F determines the direction of transfer
-in Hfr1 F is integrated between the leu and the azi gene
-genes are transferred beginning with leu
-in Hfr5 F is integrated between thi and his
-F has the opposite orientation in this chromosome so the genes are transferred beginning with thi
-*refer to slide 32 for images

Question 23

What is extrachromosomal DNA?

Answer 23

-plasmids and episomes
-plasmid is an extra-chromosomal circular piece of DNA that can replicate independently of the chromosome
-plasmids are not required for the survival of the host cell and serve as auxiliary genetic elements
-plasmids and episomes in E,coli
-F factor (Episome; Fertility Factor)
-an episome is a genetic element that is not essential to the host and that can either replicate autonomously or be integrated into the bacterial chromosome
-can benefit the population

Question 24

How does transduction occur?

Answer 24

-bacteriophages hijack bacterial chromosome genes during the process of phage assembly
During infection, the phage can donate the hijacked genes to another cell
-bacterial chromosome is fragmenetd and soe of thebacterial genes become incorporated inot a few phages
-cell lysis relaseases transducing phages
-if the phage transfers bacterial genes to another bacterium, recombination may take place and produce a transduced bacterial cell
Genes located close to another are more likely to be cotransduced, so the rate of cotransduction is inversely proportional to the disitance between genes

Question 25

What are the key points of the parasexual processes?

Answer 25

-transformation involves the upatke of free DNA by bacteria
-conjugation occurs when a donor cell makes contatc with a arecipient cell and then transfers DNA to the recipoent cell
-transduction occurs when a virus carries bacterial genes from a donor cell to a recipient cell