Lecture 6: Genetic Analysis and Mapping in Bacteria and Bacteriophages

Question 1

What are the benefits of using microorganisms in research?

Answer 1

• Bacteria and bacteriophages essential in genetic study
• Bacteria and virus research is valuable due to:
  - Exhibit extremely short reproductive cycles
  - Can be studied in pure culture

Question 2

Define Adaptation hypothesis

Answer 2

• Interaction of bacteriophage and bacterium is essential to acquisition of immunity to phage
• Exposure to the phage “induces” resistance in the bacteria

Question 3

Define Spontaneous mutation

Answer 3

• Considered primary source of genetic variation in bacteria
• Occurs in the presence or absence of bacteriophage T1
• Explains origin of resistance in E. coli

Question 4

Explain Prototroph

Answer 4

• Wild Type
• Can grow on minimal medium
• Can synthesize all essential organic compounds (human body can not)

Question 5

Explain Auxotroph

Answer 5

• Mutant
• Needs complete medium
• Has lost ability, via mutation, to synthesize essential compounds

Question 6

At what stage would you expect to see growth?

Answer 6

• LOG PHASE (EXPONENTIAL GROWTH) shown here!
• Lag: not as active
• Stationary phase: Equally dying

Question 7

Explain Genetic recombination

Answer 7

• Provides basis for development of chromosome mapping methodology
• Genetic information is transferred
• Results in altered genotype

Question 8

Explain Vertical Gene transfer

Answer 8

• Transfer of genetic information between members of SAME species
• Parent —> Offspring

Question 9

Explain Horizontal Gene transfer

Answer 9

• Transfer of genetic information between related but distinct species
• Plays significant role in evolution of bacteria
• SAME GENERATION

Question 10

T/F: A bacterial cell WIHTOUT a fertility factor can NOT donate DNA during conjugation

Answer 10

TRUE! Fertility factor (plasmid) must be present for conjugation to occur

Question 11

Explain F Factor (Fertility factor)

Answer 11

• Unidirectional transfer of genetic material
• F+ cells serve as D N A donors
• F− cells are the recipients
• F+ cells contain fertility factor
• Confers ability to donate D N A during conjugation (has pili)

Question 12

Explain Conjugation in bacteria

Answer 12

• Bacterial sex
• Genetic information from one bacterium is transferred
  to another
• Recombines at independent locations to become wild-
  type cells
• Prototrophs result from two auxotrophs

Question 13

Is cell-to-cell contact essential for chromosome transfer?

Answer 13

YES! Especially for the transfer of the F-factor!

Question 14

Understand how Conjugation with Pilus occurs

Answer 14

• Note: E. coli may or may NOT contain the F factor
• If F factor is present:
  
  • Cell forms sex pilus - serves as a donor of genetic information
  • Copy of F factor is transferred from F+ cell to the F- recipient, converting the recipient to the F+ state

Question 15

What is Hfr (high-frequency recombination)

Answer 15

• Hfr (high-frequency recombination)
  * Special class of F + cells
• Hfr strain donates genetic information to F− cell
  * Recipient does not become F +
  * F + × F− → recipient becomes F + (low rate of recombination)
  * H f r × F− → recipient remains F− (high rate of recombination)

Question 16

What is Time Mapping?

Answer 16

• Chromosome of Hfr strain transferred linearly
• Gene ORDER and DISTANCE between genes can be PREDICTED served as basis for first genetic map of E. coli

Question 17

Gene transfer by Hfr strains led to the understanding that E. coli chromosome is _______

Answer 17

CIRCULAR

Question 18

The first part of the donor chromosome to enter the
recipient is known as _____

Answer 18

The Origin

aka The point of integration of the F factor into the chromosome

Question 19

Will Hfr always give you an F plasmid?

Answer 19

It depends.

Question 20

Explain Plasmids

Answer 20

• Double-stranded closed CIRCULAR extrachromosomal function of DNA
• Exist in multiple copies in cytoplasm
• Contain one or more genes
• F factor can integrate into chromosome
• Replicate independently of the bacterial chromosome

Question 21

What two components do R plasmids have?

Note: high copy # meaning they are extremely present!

Answer 21

1. RTF - Resistance transfer factor
2. r-determinants

Question 22

Define RTF - Resistance Transfer Factor

Answer 22

Encodes genetic information for transfer between plasmids

Question 23

Define r-determinants

Answer 23

Confer antibiotic resistance

Question 24

Explain Col plasmids

Answer 24

• ColE1 derived from E. coli
• Encode colicins: proteins toxic to bacterial strains that do NOT harbor same plasmid
• Kill neighboring bacteria
• Not transmissible to other cells (R plasmids are)

Question 25

Define colicins

Answer 25

Proteins toxic to bacterial strains that do NOT harbor same plasmid

Question 26

Compare Endo and Exo-nuclease

Answer 26

- Endonucleases: Cut nucleotides found within the DNA strand. - Exonuclease: Cuts nucleotides found on DNA at the 3' or the 5' ends.

Question 27

Define Transformation

Answer 27

- Small pieces of extracellular DNA are taken up by bacterial cell - Integrated stably into the chromosome

Question 28

What are the 2 steps of transformation

Answer 28

- Entry of foreign DNA into recipient cell - Recombination between foreign DNA and homologous region in recipient chromosome

Question 29

What are the 2 outcomes of transformation

Answer 29

- Completion of both steps required for genetic recombination - First step alone results in additional foreign DNA to cytoplasm but NOT chromosome (Known as EPISOMAL EXPRESSION)

Question 30

Explain Heteroduplex

Answer 30

- Recombinant region that holds one host strand and one mutant strand - Strands are from different sources - Contains mismatch of base pairs

Question 31

Define Cotransformation

Answer 31

- Simultaneous transfer of genes - Genes are close enough to be linked - Are carried on single segment of 10,000-20,000 nucleotide pairs - Frequency of two unlinked genes transforming simultaneously lower than that of linked genes

Question 32

Define Bacteriophages

Answer 32

- Viruses that have bacteria as their host - Reproduce via bacterial genetic recombination mechanism: transduction

Question 33

Explain the structure of Bacteriophage T4

Answer 33

- Head of virus: DNA contained in icosahedral protein coat - Tail: Contractile sheath surrounding central core - Tail fibers contain binding sites that recognize bacterial outer surface

Question 34

Understand the Phage Life Cycle

Answer 34

- Phage absorbed to bacterial host cell - Phage DNA injected; host DNA degraded - Phage DNA replicated and phage protein components made - Mature phages assembled - Host cell lysed; phage released (Lytic viruses due to immediate reaction!)

Question 35

T/F: A lot of viruses are host specific?

Answer 35

TRUE! An e-coli virus only infects E. coli

Question 36

Phages exist in two forms. Name them!

Answer 36

1. Lytic (Virulent) 2. Lysogenic (Temperate)

Question 37

Define Plaque Assay

Answer 37

- Determines number of phages produced after infecting bacteria - Entails serial dilutions of virally infected bacteria - Counting plaques (areas clear of bacteria) on plates determines number of phages in original culture

Question 38

Define Lysogeny

Answer 38

- Phage D N A integrates into bacterial chromosome—coexists - Replicated along with the chromosome - Passed to daughter cells - No lysis of host cell

Question 39

Define Temperate Phages

Answer 39

Can either lyse cell or behave as prophage

Question 40

Define Virulent Phage

Answer 40

Can only lyse cell

Question 41

Define Prophage

Answer 41

- Viral D N A integrated into bacterial chromosome - In lysogenic stage: Capable of being lysed as a result of induced viral reproduction - A bacterium harboring a prophage has been lysogenized

Question 42

The viral DNA is classified as an ______

Answer 42

- Episome - A genetic molecule that can replicate either in the cytoplasm of a cell or as part of its chromosome

Question 43

Define Transduction

Answer 43

Bacterial recombination mediated by bacteriophages

Question 44

Explain the Lederberg-Zinder experiment

Answer 44

- Used Salmonella (auxotrophic strains) - Led to discovery of phage transduction in bacteria

Question 45

Define Generalized Transduction

Answer 45

Process of transduction where bacterial recombination is mediated by bacteriophage

Question 46

Define Cotransduction

Answer 46

- Two genes are close enough to be transduced simultaneously - Two independent transduction events may occur if genes are not close enough

Question 47

T/F: All Lysogenic phages will eventually become a Lytic phage

Answer 47

TRUE!!! If only a lytic phage, will not become a lysogenic virus

Question 48

Bacteriophage Mutations affect what?

Answer 48

Plaque morphology

Question 49

Define Mixed Infection Experiments

Answer 49

* Two distinct mutant strains simultaneously infected bacteria * Viral particles used exceeded bacterial cells to ensure simultaneous infection * Demonstrated intergenic (two loci) recombination occurs in bacteriophages

Question 50

Define Complementation

Answer 50

* During simultaneous infection, mutant strains give each other lacking genes * Restores wild type

Question 51

Define Complementation groups (cistrons)

Answer 51

* Failed to complement each other * Successfully complemented each other

Question 52

Define Cistrons

Answer 52

* Smallest functional genetic unit * Describes complementation group * Represents a gene

Question 53

Define Recombinational Analysis

Answer 53

* The percentage of recombinants can be determined by counting the plaques at the appropriate dilution * The frequency of recombination is an estimate of the distance between the two mutations within the cistron.