Topic E - Genetic Analysis of T4

T4 Phage

-Complex protein coat

-dsDNA containing hydroxymethyl-cytosine (protects against bacterial immune system)

-Lytic bacteria phage

Steps in Genetic Analysis

1. Generation of Mutation

2. Identification of Mutant Phage

3. Preparation of Pure Stocks of Mutant Phage

4. Virus Crosses

Generation of Mutation

Treatment with a mutagen/hope for spontaneous mutation

identification of Mutant Phage

Screen or select for:

-Plaque morphology

-Growth/lack of growth (may require replica plating)

Preparation of Pure Stocks

Start from single, isolated plaque of mutant phage

-Infect culture of sensitive bacteria under permissive conditions → pure lysate/stock

Virus Crosses

Co-infect bacterial culture with 2 different mutant phage

-Use high MOI so each bacterial cell infected by both types of phages

-Phage genes expressed, DNA can recombine

Uses of Viral Crosses

1. Complementation Analysis - Tells us number of different genes affected by mutation

2. Genetic Linkage Mapping - Intergenic recombination, tells us where genes located relative to one another

3. Fine-Structure Mapping - Intragenic recombination, tells us how mutations are distributed within a single gene

Complementation Analysis

1. Co-Infect host with 2 different mutant phage that have the same phenotype

2. Plate under restrictive conditions:

• Either restrictive host or temperature-sensitive

• Mutant phage alone can’t replicate

3. Examine plates for wildtype phenotype

• Plaque formation in restrictive condition

Interpreting Complementation Results

Analysis always done under restrictive conditions

Identification of T4 rII Mutant Genes

T4 rII mutants form large plaques since Anti-Holin is not produced (no lysis inhibition at plaque edge, results in very large plaques)

Complementation Groups in T4 rII Mutants

Group 1: rII 1, rII 4

Group 2: rII 2, rII 3

-Therefore a total of two complementation groups

Constructing a Gene Map - Intergenic Recombination

Determines gene order in genome and distance between genes

-Requires most phage genes have been identified

Constructive a Gene Map - Two-Factor Cross

1. Select 2 single mutants in different complementation groups (2 different genes)

2. Co-infect bacteria at high MOI with 2 mutant phage strains (ensure co-infection)

3. Plate progeny under permissive conditions

• Count total number of progeny

4. Replicate plate to restrictive conditions

• Count WT recombinant progeny

• Determine total number of recombinants

Double mutants not seen in restrictive conditions: so WT = 1/2 of recombinants

5. Calculate recombination frequency