Biochemistry 3107 - Fall 2003

Introduction to Recombination

Recombination

Recombination is a process or set of processes by which DNA molecules interact with one another to bring about a rearrangement of the genetic information or content in an organism.

In eukaryotic systems, you will be familiar with recombination as the process that is responsible for crossing-over during meiosis. Crossing-over has been well-documented genetically and is used to map the relative locations of genes on a chromosome. Implicit in the models and diagrams of meiosis that you have seen before is the idea that crossing over - and recombination - must involve the breakage and rejoining of DNA molecules. Crossing-over can also be seen physically by the visible occurence of chiasmata in meiotic tetrads. Recombination, as we will see later, is also responsible for the generation of antibody diversity.

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Examples of recombination in prokaryotic systems are (i) integration of the bacteriophage lambda prophage, (ii) recombination of bacterial DNA following conjugation between bacteria, and (iii) formation of plasmid multimers.

 

We can distinguish 4 general types of recombination:

Homologous genetic recombination

Also known as general recombination or general homologous recombination this is the exchange of genetic material between two molecules that share a large degree of identity with one another. This is the type of recombination that is required during meiotic crossing over, for bacteriophage recombination, for recombination following bacterial conjugation, and during the formation of plasmid multimers

 

Site-specific recombination

As the name implies, this type of recombination involves the exchange of genetic material at very specific sites only. Examples include the integration of bacteriophage lambda into the host chromosome to form the prophage and the rearrangement of chromosomal DNA prior to expressing antibody genes.

 

DNA transposition

Barbara McClintock (see also a brief bio at Cold Spring Harbor; and an autobiography at the Nobel Prize site) spent many years patiently studying the behaviour of unusual genetic elements in maize. She concluded that these elements were, in fact, mobile. Her work, all the more amazing because much of it was carried out before the structure of DNA was solved, was largely ignored until the mid 1970s when similar elements were discovered in bacteria.

 

Illegitimate recombination

There are a number of other genetic exchanges which do not fall into any of the above classes - hence their name: illegitimate recombination.

 

In this course, we will cover general homologous recombination, site-specific recombination, and transposition. We will not cover illegitimate recombination.

 

Recombination in bacteria

Recombination in a bacterial system was first demonstrated independently by Al Hershey and Max Delbrück in 1947. They studied the infection of E. coli with bacteriophage. If an E. coli cell was infected at the same time with two genetically different bacteriophage, the resulting phage population included recombinant phage types as well as the original parental phage types.

The ease with which large numbers of phage particles can be handled facilitated the discovery and characterization of mutants that were easily scored. Al recognized that the high infectivity of phage and the proportionality of plaque count to volume of suspension assayed allowed for quantification of mutation far exceeding that possible in most other viral systems. Al measured mutation rates, both forward and back, and demonstrated the mutational independence of r (rapid lysis) and h (host range). He succeeded also in showing (in parallel with Delbrück) that these mutationally independent factors could recombine when two genotypes were grown together in the same host cells (1946, 1947). Thus phage genetics was born as a field of study, and it became conceivable that not only could the basic question of biological replication be addressed with phage but so also could phenomena embraced by the term "Morgan-Mendelism."

FROM: A biographical memoir of Alfred Day Hershey (December 4, 1908 - May 22, 1997) by Franklin W. Stahl

In the classical experiment of this type, performed by Al Hershey, E. coli was infected with two strains of bacteriophage T2. One strain is h^-r⁺; the other is h⁺r^-.

The h locus determines whether the phage can grow on a particular strain of E. coli:

• phage that are h^- can infect the strain;
  
• phage that are h⁺ cannot.

The r locus is a gene that determines whether the phage will lyse the host cells rapidly or slowly:

• phage that are r^- will lyse the host cells rapidly;
  
• phage that are r⁺ will lyse the host cell slowly.

 

In addition, two strains of E. coli were used in the experiment:

• strain 1 supports growth of h^- phage but not h⁺ phage;
  
• strain 2 supports the growth of both phage.

 

In the experiment, bacteriophage are plated on a lawn of bacteria that consists of a mixture of both strains of E. coli. If a phage can infect both strains of bacteria (i.e. if it is h^-) then the resulting plaque will be clear. If the phage can infect only one of the two strains of bacteria (i.e. if it is h⁺) then the resulting plaque will be turbid because the non-infected bacteria will be growing.

When the experiment is performed, four types of plaque were seen:

phenotype	inferred genotype
Clear and small	h-r+
Cloudy and large	h+r-
Cloudy and small	h+r+
Clear and large	h-r-

 

Most of the plaques correspond to the parental phenotypes but a significant number have the recombinant phenotypes.

Note that, when the progeny phage were used to reinfect E. coli so as to examine their phenotype, a low but definite percentage of the resulting plaques were found to contain two different types of phage although only one type had been expected. This implies that some of the progeny phage were not genetically homogeneous.

This observation can be explained by models of recombination that allow for heteroduplex forms to be generated.

Al Hershey and Max Delbrück shared the 1969 Nobel prize in Medicine & Physiology with Salvador Luria for their discoveries concerning "the replication mechanism and the genetic structure of viruses" [press release].

The Meselson - Weigle Experiment

In the simplest sense, recombination is an exchange of both strands between two DNA molecules:

Note: each line in the above cartoon figure represents one strand of a DNA double helix.

This representation implies that both strands of each molecule must be broken and then rejoined. This was first demonstrated by an experiment performed by Matt Meselson and Jean Weigle in 1961.

Meselson and Weigle infected E. coli cells at the same time with phage from two different stocks of bacteriophage lambda. One stock had been prepared by growing the bacteriophage lambda c^-mi^- in cells grown in medium containing heavy isotopes of carbon (¹³C) and nitrogen (¹⁵N). The other stock had been prepared by growing bacteriophage lambda c⁺mi⁺ in medium containing light isotopes of carbon and nitrogen.

Note: each line in the above figure represents a phage chromosome, i.e. a double helical DNA molecule.

After infection, the progeny phage were isolated and banded on a CsCl gradient. A broad band of phage particles were found on the gradient. Nonrecombinant phage were found, as expected, at two well-defined densities corresponding to the parental light and heavy phages. Recombinant phage were found - surprisingly - at all intermediate densities between these two.

NOTE: The above image may be restricted to users from licensed or registered sites.

They also followed the course of the infection using two genetic markers, c and mi, which were located near one end of the lambda chromosome.

Note: each line in the above figure represents a phage chromosome, i.e. a double helical DNA molecule.

When the phenotypes of the intermediate density phage particles were analyzed, recombinant phage that were c^-mi⁺ were found near the band of "heavy" phage while recombinant phage that were c⁺mi^- were found near the band of "light" non-recombinant phage.

These results can only be explained if recombination between the two parental phage involves breakage and rejoining of both DNA strands (as shown above).