INTRAGENIC CROSSING-OVER & COMPLIMENTATION

Submitted to: Submitted by:Dr. Bharat singh Deepti barange B.tech biotech 4th sem

WHAT IS INTRAGENIC CROSSING OVER? This simply means recombination within a

gene. In early 1950s Seymour Benzer undertook a

detailed examination of a single locus, rII,in phage T4

He successfully designed experiments to recover the extremely rare genetic recombinations arising as a result of intragenic exchange.

He demonstrated such recombination occurs between DNA of individual bacteriophages during simultaneous infection of the host bacterium E.coli

His work is described as fine structure analysis due to extremely detailed information provided from his analysis

WHAT ARE PLAQUES?A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages

Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaquesThus, plaques can be viewed as traits of

bacteriophages Plaques are visible with the naked eye

So mutations affecting them lend themselves to easier genetic analysis

An example is a rapid-lysis mutant of bacteriophage T4, which forms unusually large plaquesThis mutant lyses bacterial cells more rapidly

than do the wild-type phages Rapid-lysis mutant forms large, clearly defined

plaques Wild-type phages produce smaller, fuzzy-edged

plaques

Benzer’s fine-structure mapping of phage T4 used similar experiments involving the rII gene. a. Different rII mutations of T4 were used, each with the

characteristic large clear plaques and limited host range.

b. T4 with the wild-type r+ gene infects E. coil strains B and K12(λ). But For rII T4(mutant), strain B is permissive but K12(λ) is nonpermissive.

In E. coli B rII phages produced unusually large plaques that had poor

yields of bacteriophages The bacterium lyses so quickly that it does not have time to

produce many new phages

In E. coli K12S rII phages produced normal plaques that gave good yields

of phages

In E. coli K12(λ)has phage lambda DNA integrated into its chromosome) rII phages were not able to produce plaques at all

BENZER’S GENERAL PROCEDURE FOR DETERMINING THE NUMBER OF R+ RECOMBINANTS FROM A CROSS INVOLVING TWO RII MUTANTS OF T4

COMPLIMENTATION

Benzer collected many rII mutant strains that can form large plaques in E. coli B & none in E. coli K12(λ)

But, are the mutations in the same gene or in different genes?

To answer this question, he conducted complementation experiments

For the production of phenotype the presence of both wild type genes is required. So if the mutation is present on two different genes of the parents the progeny will still have one wild type gene from each parent, i.e. the genes will compliment each other while in the second case mutation is present on one gene in both parents , i.e. progeny will have only one wild type gene which will be insufficient to give phenotype.

Benzer carefully considered the pattern of complementation & noncomplementationHe determined that the rII mutations occurred in

two different genes, which were termed rIIA & rIIB

Benzer coined the term cistron to refer to the smallest genetic unit that gives a negative complementation testSo, if two mutations occur in the same cistron,

they cannot complement each other

A cistron is equivalent to a gene

At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred

DESCRIBES THE GENERAL STRATEGY FOR INTRAGENIC MAPPING OF RII PHAGE MUTATIONS

THE DATA FROM FIGURE CAN BE USED TO ESTIMATE THE DISTANCE BETWEEN THE TWO MUTATIONS IN THE SAME GENE The phage preparation used to infect E. coli B was diluted by

108 (1:100,000,000) 1 ml of this dilution was used & 66 plaques were produced Therefore, the total number of phages in the original

preparation is 66 X 108 = 6.6 X 109 or 6.6 billion phages per milliliter

The phage preparation used to infect E. coli k12(λ) was diluted by 106 (1:1,000,000)

1 ml of this dilution was used & 11 plaques were produced Therefore, the total number of wild-type phages is 11 X 106

In this experiment, the intragenic recombination produces an equal number of recombinants Wild-type phages & double mutant phages

However, only the wild-type phages are detected in the infection of E. coli k12(λ) Therefore, the total number of recombinants is the number

of wild-type phages multiplied by two

or 11 million phages per milliliter

DELETION MAPPING Benzer used deletion mapping to localize many rII

mutations to a fairly short region in gene A or gene B Let’s suppose that the goal is to know the

approximate location of an rII mutation, such as r103 E. coli k12(λ) is coinfected with r103 & a deletion

strain (deletion strains don't have the gene )

If the deleted region includes the same region that contains the r103 mutationNo intragenic wild-type recombinants are

producedTherefore, plaques will not be formed

If the deleted region does not overlap with the r103 mutation Intragenic wild-type recombinants can be

producedAnd plaques will be formed

On the basis of these studies he mapped the rII gene as:

Contain many mutations at exactly the same site within

the gene

Thank you !!