T4 rII system
Encyclopedia
The T4 rII system is an experimental system
developed in the 1950s by Seymour Benzer
for studying the substructure of the gene
. The experimental system is based on genetic crosses of different mutant
strains
of bacteriophage T4, a virus that infects the bacteria E. coli.
rather than the smaller plaques characteristic of the wild type
virus. Through genetic mapping, these phage workers had identified specific regions in the T4 chromosome, called the rI, rII, and rIII loci
, associated with the r mutants. In 1952, while performing experiments with rII mutants, Seymour Benzer found a strain that did not behave normally. By 1953, after the publication of Watson and Crick
's proposed structure of DNA
, Benzer hit on the idea that the apparently defective r mutants might have been the result of crossing two different rII mutants, each of which had part of the rII gene intact, so that hybrid strain did not exhibit the r phenotype at all because it combined the intact parts of the rII gene.
From there, Benzer saw that it would be possible to generate many independent r mutants, and by measuring the recombination frequency between different r strains, he could map the substructure of a single gene. Although the chance of successful recombination for any given virus was very small, a single petri dish could be the basis for millions of trials at once. They could be screened easily by using a specific strain of E. coli, known as K12 (λ), that was susceptible to wild type T4 but not to r mutants.
Benzer's concept was quite controversial within classical genetic thought, in which each gene is treated as a singular point along a chromosome, not a divisible stretch of nucleic acids (as implied by the work of Watson and Crick). Initially, Max Delbrück
—a respected phage geneticists and leader of the so-called phage group
of which Benzer was a part—found Benzer's idea outrageous.
s".
Benzer identified a number of different types of r mutants. Some he classified as deletions, others as point mutation
s. By various crosses of the many different strains exhibited deletions and point mutations, Benzer located each point mutation into a sub-region of one of the cistrons, and ordered the point mutations within that sub-region. Benzer also proposed missense and nonsense mutation
s from his rII studies. The T4 rII system enabled Benzer to identify recombination frequencies as low as .02%, much lower than in typical genetics experiments. This was equivalent to detecting recombination between only one or two base pairs.
and others used one of the peculiar r mutants Benzer had found (a deletion that fused the A and B cistrons of rII) to demonstrate the triplet nature of the genetic code
.
Experimental system
In scientific research, an experimental system is the physical, technical and procedural basis for an experiment or series of experiments. Historian of science Hans-Jörg Rheinberger defines an experimental system as: "A basic unit of experimental activity combining local, technical, instrumental,...
developed in the 1950s by Seymour Benzer
Seymour Benzer
Seymour Benzer was an American physicist, molecular biologist and behavioral geneticist. His career began during the molecular biology revolution of the 1950s, and he eventually rose to prominence in the fields of molecular and behavioral genetics. He led a productive genetics research lab both at...
for studying the substructure of the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
. The experimental system is based on genetic crosses of different mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
strains
Strain (biology)
In biology, a strain is a low-level taxonomic rank used in three related ways.-Microbiology and virology:A strain is a genetic variant or subtype of a micro-organism . For example, a "flu strain" is a certain biological form of the influenza or "flu" virus...
of bacteriophage T4, a virus that infects the bacteria E. coli.
Origin
One type of mutation in T4 bacteriophage identified by researchers in phage genetics by the 1950s was known r (for rapid), which caused the phage to destroy bacteria more quickly than normal. These could be spotted easily because they would produce larger plaquesViral plaque
A viral plaque is a visible structure formed within a cell culture, such as bacterial cultures within some nutrient medium . The bacteriophage viruses replicate and spread, thus generating regions of cell destructions known as plaques....
rather than the smaller plaques characteristic of the wild type
Wild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
virus. Through genetic mapping, these phage workers had identified specific regions in the T4 chromosome, called the rI, rII, and rIII loci
Locus (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
, associated with the r mutants. In 1952, while performing experiments with rII mutants, Seymour Benzer found a strain that did not behave normally. By 1953, after the publication of Watson and Crick
Watson and Crick
James D. Watson and Francis Crick were the two co-discoverers of the structure of DNA in 1953. They used x-ray diffraction data collected by Rosalind Franklin and proposed the double helix or spiral staircase structure of the DNA molecule...
's proposed structure of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, Benzer hit on the idea that the apparently defective r mutants might have been the result of crossing two different rII mutants, each of which had part of the rII gene intact, so that hybrid strain did not exhibit the r phenotype at all because it combined the intact parts of the rII gene.
From there, Benzer saw that it would be possible to generate many independent r mutants, and by measuring the recombination frequency between different r strains, he could map the substructure of a single gene. Although the chance of successful recombination for any given virus was very small, a single petri dish could be the basis for millions of trials at once. They could be screened easily by using a specific strain of E. coli, known as K12 (λ), that was susceptible to wild type T4 but not to r mutants.
Benzer's concept was quite controversial within classical genetic thought, in which each gene is treated as a singular point along a chromosome, not a divisible stretch of nucleic acids (as implied by the work of Watson and Crick). Initially, Max Delbrück
Max Delbrück
Max Ludwig Henning Delbrück was a German-American biophysicist and Nobel laureate.-Biography:Delbrück was born in Berlin, German Empire...
—a respected phage geneticists and leader of the so-called phage group
Phage group
The phage group was an informal network of biologists centered around Max Delbrück that contributed heavily to bacterial genetics and the origins of molecular biology in the mid-20th century. The phage group takes its name from bacteriophages, the bacteria-infecting viruses that group used as...
of which Benzer was a part—found Benzer's idea outrageous.
Benzer's work
Beginning in 1954, Benzer put the T4 rII system to use, creating and crossing hundreds of r mutants and developing an increasingly detailed map of the structure of the rII gene. In his early work, he identified two separate but very close loci within the rII region, which he suggested were nucleotide sequences that encoded different polypeptides; he called these "cistronCistron
A cistron is a gene. The term cistron is used to emphasize that genes exhibit a specific behavior in a cis-trans test; distinct positions within a genome are cistronic when mutations at the loci exhibit the same simple Mendelian inheritance as would mutations at a single locus.For example,...
s".
Benzer identified a number of different types of r mutants. Some he classified as deletions, others as point mutation
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
s. By various crosses of the many different strains exhibited deletions and point mutations, Benzer located each point mutation into a sub-region of one of the cistrons, and ordered the point mutations within that sub-region. Benzer also proposed missense and nonsense mutation
Nonsense mutation
In genetics, a nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. It differs from a missense mutation, which is a point mutation...
s from his rII studies. The T4 rII system enabled Benzer to identify recombination frequencies as low as .02%, much lower than in typical genetics experiments. This was equivalent to detecting recombination between only one or two base pairs.
Work by others
After Benzer demonstrated the power of the T4 rII system for exploring the fine structure of the gene, others adapted the system to explore related problems. For example, Francis CrickFrancis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
and others used one of the peculiar r mutants Benzer had found (a deletion that fused the A and B cistrons of rII) to demonstrate the triplet nature of the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
.