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Gene Transfer in Bacteria and Bacteriophage. Using Gene Transfer Between Bacteria As a Means for Studying Bacterial Genes. Types of Traits Studied. For bacteria -need for nutrients prototropic: can grow on minimal medium

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gene transfer in bacteria and bacteriophage

Gene Transfer in Bacteria and Bacteriophage

Using Gene Transfer Between Bacteria As a Means for Studying Bacterial Genes

types of traits studied
Types of Traits Studied
  • For bacteria

-need for nutrients

prototropic: can grow on minimal medium

auxotropic: must have specific nutrients added to medium

-morphology of colonies

-resistance/sensitivity to antibiotics

  • For bacteriophage

-host range (ability to infect specific bacteria)

-appearance of plaques(shows growth)

testing for nutritional requirements
Testing for Nutritional Requirements

Replica plating transfers the pattern of bacterial colonies to test plates.

dna of prokaryotic cells
DNA of Prokaryotic Cells
  • Bacterial cells have a single, circular chromosome and therefore have one copy of each gene.
  • Partial diploids (merozygotes) can be formed by the introduction of genetic material from another cell.
gene transfer processes for bacteria and their viruses
Gene Transfer Processes for Bacteria and Their Viruses
  • Conjugation
  • Transformation
  • Transduction
  • Infection with bacteriophage
gene transfer processes for bacteria and their viruses6
Gene Transfer Processes for Bacteria and Their Viruses

1. Conjugation

Transfer of DNA from one bacterial cell to another

Donor cell (F+ or Hfr) transfers DNA to recipient cell (F-)

genetic analyses using conjugation
Genetic Analyses Using Conjugation
  • Determining linkage from interrupted mating experiments
  • Determining gene order from gradient of transfer
  • Higher-resolution mapping by recombination frequency
genetic analyses using conjugation9
Genetic Analyses Using Conjugation
  • Determining linkage from interrupted mating experiments

Combine Hfr strain (Strs) and F- strain.

Remove samples at specific time intervals.

Use blender to disrupt mating.

Plate on streptomycin to kill donor cells.

Test recipient cells for genes from Hfr strain.

genetic analyses using conjugation10
Genetic Analyses Using Conjugation

A. Determining linkage from interrupted mating experiments

Problem 1, page 2-4

Three Hfr strains for E. coli are mated individually with an auxotrophic F- strain using an interrupted mating procedure. Approximate times of entry of each gene are listed below. Determine the map of the E. coli chromosome and show the orientation of the F plasmid in each Hfr strain.

genetic analyses using conjugation11
Genetic Analyses Using Conjugation

A. Determining linkage from interrupted mating experiments

Problem 1, page 2-4

genetic analyses using conjugation12
Genetic Analyses Using Conjugation

A. Determining linkage from interrupted mating experiments

Problem 1, page 2-4

genetic analyses using conjugation13
Genetic Analyses Using Conjugation

B. Determining gene order from gradient of transfer

Combine Hfr and F- strains.

Allow for natural disruption of conjugated pairs.

Select for earliest transferred marker.

Test for markers transferred later in conjugation.

genetic analyses using conjugation14
Genetic Analyses Using Conjugation

B. Determining gene order from gradient of transfer

Problem 2, page 2-4

An Hfr strain donates the genes xyl+ pro+lac+ and gal+ to an F- strain. Recombinants are selected forgal+. Tests are done to determine the presence of the other three genes in the gal+ recombinants. What is the gene order?

genetic analyses using conjugation15
Genetic Analyses Using Conjugation

2. Determining gene order from gradient of transfer

Problem 2, page 2-4

Select for gal+

Test for lac+, pro+, xyl+

genetic analyses using conjugation17
Genetic Analyses Using Conjugation

C. Higher-resolution mapping by recombination frequency

Combine Hfr and F- strains.

Allow for natural disruption of conjugated pairs.

Select for marker that enters LAST.

Test for unselected markers.

genetic analyses using conjugation18
Genetic Analyses Using Conjugation

C. Higher-resolution mapping by recombination frequency

Problem 3, page 2-4

An Hfr strain that is met+arg+leu+strs is conjugated with an F- strain that is met-arg-leu-strr. Interrupted mating studies show that leu+ enters last. Recombinants that are leu+strr are selected and then tested for the presence of met+ and arg+. The following numbers of bacteria are found for each of the genotypes listed below. Determine the gene order and the distances between the genes in map units.

genetic analyses using conjugation19
Genetic Analyses Using Conjugation

C. Higher-resolution mapping by recombination frequency

Problem 3, page 2-4

Select for leu+Test for met+, arg+

genetic analyses using conjugation20
Genetic Analyses Using Conjugation

C. Higher-resolution mapping by recombination frequency

Problem 3, page 2-4

leu+ met+ arg+

Hfr

met-

arg-

leu-

F-

genetic analyses using conjugation21
Genetic Analyses Using Conjugation

Problem 3, page 2-4

Smallest number of offspring represents 4 crossovers, identifies middle gene.

Genotype will be leu+ met- arg+.

leu+ met+ arg+

Hfr

met-

arg-

leu-

F-

genetic analyses using conjugation22
Genetic Analyses Using Conjugation

Problem 3, page 2-4

Recombination between leu and met gives

leu+ met- arg- offspring.

leu+ met+ arg+

Hfr

met-

arg-

leu-

F-

genetic analyses using conjugation23
Genetic Analyses Using Conjugation

Problem 3, page 2-4

Recombination between met and arg gives leu+ met+ arg- offspring.

leu+ met+ arg+

Hfr

met-

arg-

leu-

F-

gene transfer processes for bacteria and their viruses25
Gene Transfer Processes for Bacteria and Their Viruses

2. Transformation

DNA taken up from external environment

genetic analysis using transformation
Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

Transform bacteria with DNA containing two markers (eg. his-, met-) in addition to penicillin sensitivity.

Select transformants on minimal medium + penicillin to kill non-transformants.

Plate survivors on complete medium to test for his-, met-.

genetic analysis using transformation27
Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

Problem 4, page 2-5

DNA is isolated from E. coli strain A (his- met- pens) and used to transform strain B (his+ met+ pens). Transformants are selected on minimal medium + penicillin to kill his+ met+ cells and survivors are plated on complete medium. The classes and numbers of cells obtained are listed below. Determine the recombination frequency between the his and met genes.

genetic analysis using transformation28
Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

Problem 4, page 2-5

Rf = number of single transformants

total number of transformants

genetic analysis using transformation29

his- met-

met+

his+

Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

genetic analysis using transformation30
Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

Single transformants, his- met+ and his+ met-,

represent crossovers between the genes.

his- met-

his- met-

met+

met+

his+

his+

genetic analysis using transformation31
Genetic Analysis Using Transformation

Determining genetic distance with transformation mapping

Problem 4, page 2-5

gene transfer processes for bacteria and their viruses32
Gene Transfer Processes for Bacteria and Their Viruses

3. Transduction Transfer of bacterial genes with a bacteriophage

genetic analysis using transduction
Genetic Analysis Using Transduction

Determining cotransduction frequency with three-factor transduction.

Cotransduction frequency = tendency for genes to be transferred together on same piece of transducing DNA

genetic analysis using transduction35
Genetic Analysis Using Transduction

Three-factor transduction:

Transducingbacteriophage are used to transfer DNA with three markers to bacterial cells.

Bacteria are selected for one of the markers and tested for the presence of the other two markers.

Gene order and cotransduction frequency can be determined.

gene transfer processes for bacteria and their viruses36
Gene Transfer Processes for Bacteria and Their Viruses

Three-factor transduction

Problem 6, Page 2-5

Transducing phages that infected an A+B+C+ cell are used to infect an A-B-C- cell. Transductants receiving the A+ marker were tested for the presence of B+ andC+. The classes and numbers of transductants observed is shown below. Determine the gene order and the cotransduction frequencies for A+ with B+ and A+ with C+.

gene transfer processes for bacteria and their viruses37
Gene Transfer Processes for Bacteria and Their Viruses

Three-factor transduction

Problem 6, Page 2-5

Select for A+Test for B+ andC+

genetic analysis using transduction38
Genetic Analysis Using Transduction

Problem 6, page 2-5

Smallest number of offspring represents 4 crossovers, identifies middle gene. Genotype will be A+ B- C+.

A+ B+ C+

B-

C-

A-

genetic analysis using transduction39
Genetic Analysis Using Transduction

Problem 6, page 2-5

Cotransduction of A and B

Cotransduction of A and C

genetic analysis using transduction40
Genetic Analysis Using Transduction

Problem 6, page 2-5

Cotransduction of A and B =

Cotransduction of A and C =

The higher the cotransduction frequency, the closer the genes are to each other.

gene transfer processes for bacteria and their viruses41
Gene Transfer Processes for Bacteria and Their Viruses

4. Infection with bacteriophage

In a mixed infection,recombination can be detected between bacteriophage carrying different genes.

gene transfer processes for bacteria and their viruses42
Gene Transfer Processes for Bacteria and Their Viruses

Infection with bacteriophage

Infect bacteria with bacteriophage of two different genotypes.

Recombination can occur between bacteriophage genes.

Determine genotypes of resulting bacteriophage.

Rf = number of recombinant plaques

total number of plaques

gene transfer processes for bacteria and their viruses43
Gene Transfer Processes for Bacteria and Their Viruses

Infection with bacteriophage

lawn of bacterial cells

Plaque for

one genotype

Plaque for

alternate

genotype

genetic analysis for infection with bacteriophage

ra-

h+

ra+

h-

Recombinant Types

Parental Types

ra- h-

ra- h+

ra+ h+

ra+ h-

Genetic Analysis for Infection With Bacteriophage

X

gene transfer processes for bacteria and their viruses45
Gene Transfer Processes for Bacteria and Their Viruses

Infection with bacteriophage

Problem 5, Page 2-5

Three different bacteriophage T2 strains carrying mutations in the r gene (ra, rband rc) were each involved in a cross r-xh+ X r+xh-, where x=a, b or c. The numbers of bacteriophage of each type are listed below. Give any one of four possible linkage maps for these genes.

gene transfer processes for bacteria and their viruses46
Gene Transfer Processes for Bacteria and Their Viruses

4. Infection with bacteriophage

Problem 5, Page 2-5

gene transfer processes for bacteria and their viruses47
Gene Transfer Processes for Bacteria and Their Viruses

Infection with bacteriophage

Rf = number of recombinant plaques

total number of plaques

gene transfer processes for bacteria and their viruses48
Gene Transfer Processes for Bacteria and Their Viruses

Infection with bacteriophage

One possible map: