Negative Regulation: The E coli lac operon The classic example of negative regulation is regulation of the e coli lac operon. which encodes the enzymes responsible for the utilization of the sugar lactose. The experiments of francois Jacob and Jacques monod and their collaborators on the regulation of the E. coli lac genes are excellent examples of the genetic anal ysis of a biological phenomenon in bacteria Although these experiments were per formed in the late 1950s only shortly after the discovery of the structure of dna and the existence of mRNA they still stand as the framework with which all other studies of gene regulation are compared
• Negative Regulation:The E. coli lac Operon • The classic example of negative regulation is regulation of the E. coli lac operon, which encodes the enzymes responsible for the utilization of the sugar lactose. The experiments of Francois Jacob and Jacques Monod and their collaborators on the regulation of the E. coli lac genes are excellent examples of the genetic analysis of a biological phenomenon in bacteria. Although these experiments were performed in the late 1950s, only shortly after the discovery of the structure of DNA and the existence of mRNA, they still stand as the framework with which all other studies of gene regulation are compared
GENETICS OF THE Lac Operon When Jacob and monod began their classic work, it was known that the enzymes of lactose metabolism are inducible in that they are expressed only when the sugar lactose is present in the medium. If no lactose is present the enzymes are not made. From the standpoint of the cell, this is a sensible strategy, since there is no point in making the enzymes for lactose utilization unless there is lactose available for use as a carbon and energy source To understand the regulation of the lactose genes, Jacob and Monod first isolated many mutations affecting lactose metabolism and regulation which fell into two fundamentally different groups. Some mutants were unable to grow with lactose as the sole carbon and energy source and so were lac Other mutants made the lactose-metabolizing enzymes whether or not lactose was present in the medium and so were constitutive mutants
• GENETICS OF THE lac OPERON • When Jacob and Monod began their classic work, it was known that the enzymes of lactose metabolism are inducible in that they are expressed only when the sugar lactose is present in the medium. If no lactose is present, the enzymes are not made. From the standpoint of the cell, this is a sensible strategy, since there is 'no point in making the enzymes for lactose utilization unless there is lactose available for use as a carbon and energy source. • To understand the regulation of the lactose genes, Jacob and Monod first isolated many mutations affecting lactose metabolism and regulation, which fell into two fundamentally different groups. Some mutants were unable to grow with lactose as the sole carbon and energy source and so were Lac- . Other mutants made the lactose-metabolizing enzymes whether or not lactose was present in the medium and so were constitutive mutants
To analyze the regulation of the lac genes, Jacob and monod needed to know which of the mutations affected trans-acting gene products-either protein or rna--involved in the regulation and how many different genes these mutations represented. They also wished to know if any of the mutations were cis acting(affecting sites on the dna involved in regulation) To answer these questions, they needed to perform complementation tests, which require that the organisms be diploid, with two copies of the genes being tested. Bacteria are normally haploid, with only one copy of each of their genes but are"partial diploids" for any genes carried on an introduced prime factor. Recall that a prime factor is a plasmid into which some of the bacterial chromosomal genes have been inserted. By introducing prime factors carrying various mutated lac genes into cells with different mutations in the chromosomal lac genes, Jacob and monod performed complementation tests on each of their lac mutations Their methods depended upon the type of mutation being tested
• To analyze the regulation of the lac genes, Jacob and Monod needed to know which of the mutations affected trans-acting gene products—either protein or RNA—involved in the regulation and how many different genes these mutations represented. They also wished to know if any of the mutations were cis acting (affecting sites on the DNA involved in regulation). • To answer these questions, they needed to perform complementation tests, which require that the organisms be diploid, with two copies of the genes being tested. Bacteria are normally haploid, with only one copy of each of their genes, but are "partial diploids" for any genes carried on an introduced prime factor. Recall that a prime factor is a plasmid into which some of the bacterial chromosomal genes have been inserted. By introducing prime factors carrying various mutated lac genes into cells with different mutations in the chromosomal lac genes, Jacob and Monod performed complementation tests on each of their lac mutations. Their methods depended upon the type of mutation being tested
CoMPLEMENTATION TESTS WITH lac MUTATIONS Whether a particular lac mutation is dominant or recessive was determined by introducing an F factor carrying the wild-type lac region into a strain with the lac mutation in the chromosome. If the partial diploid bacteria are lac- and can multiply to form colonies on minimal plates with lactose as the sole carbon and energy source the lac mutation is recessive. If the partial diploid cells are lac and cannot form colonies on lactose minimal plates, the lac mutation is dominant. Jacob and monod discovered that most lac mutations are recessive to the wild type and so presumably inactivate genes whose products are required for lactose utilization
• COMPLEMENTATION TESTS WITH lac MUTATIONS • Whether a particular lac mutation is dominant or recessive was determined by introducing an F factor carrying the wild-type lac region into a strain with the lac mutation in the chromosome. If the partial diploid bacteria are Lac- and can multiply to form colonies on minimal plates with lactose as the sole carbon and energy source, the lac mutation is recessive. If the partial diploid cells are Lac- and cannot form colonies on lactose minimal plates, the lac mutation is dominant. Jacob and Monod discovered that most lac mutations are recessive to the wild type and so presumably inactivate genes whose products are required for lactose utilization
The question of how many genes are represented by recessive lac mutations could be answered by performing pairwise complementation tests between different lac mutations. Prime factors carrying the lac region with one lac mutation were introduced into a mutant strain with another lac mutation in the chromosome. In this kind of experiment, if the partial diploid cells are Lact, the two recessive mutations can complement each other and are members of different complementation groups or genes. If the partial diploid cells are Lac, the two mutations cannot complement each other and are members of the same complementation group or gene Jacob and monod found that most of the lac mutations sorted into two different complementation groups, which they named lacz and lac r We now know of another gene laca, which was not discovered in their original selections because its product is not required for growth on lactose
• The question of how many genes are represented by recessive lac mutations could be answered by performing pairwise complementation tests between different lac mutations. Prime factors carrying the lac region with one lac mutation were introduced into a mutant strain with another lac mutation in the chromosome. In this kind of experiment, if the partial diploid cells are Lac+ , the two recessive mutations can complement each other and are members of different complementation groups or genes. If the partial diploid cells are Lac- , the two mutations cannot complement each other and are members of the same complementation group or gene. Jacob and Monod found that most of the lac mutations sorted into two different complementation groups, which they named lacZ and lacY We now know of another gene, lacA, which was not discovered in their original selections because its product is not required for growth on lactose