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lac Mutants
L lac Mutants J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0737
Lac mutants are organisms that contain mutations in some part of the lac operon or its controlling elements. Therefore, they contain some defect in the metabolism of the disaccharide lactose, or in the regulation of this metabolism, when compared with wildtype strains. Lac mutants are of historic interest because they helped to uncover the structure and regulation of the lac operon, the first operon discovered. They are also of interest because the techniques which were developed to screen or select these mutants are still used in the classroom and the laboratory. Wild-type strains of the bacterium Escherichia coli are phenotypically Lac, meaning they have the ability to use lactose as a sole source of carbon. In order to be Lac, E. coli must be able to express a functional lacZ gene, which encodes b-galactosidase, and a functional lacY gene, which encodes the lactose permease. Mutants in which either of these genes have been inactivated are said to be Lac and cannot utilize lactose. Joshua Lederberg and his associates were the first to isolate and map Lac mutants of E. coli, beginning in the 1940s. Lac mutants can be identified by their failure to grow when lactose is the sole carbon source or by the use of various types of indicator plates. Mutations in lacZ or lacY can be differentiated by a variety of techniques. For example, mutants which cannot produce the lactose permease also cannot grow on melibiose under certain conditions. Mutations are also known in the regulatory genes or regions controlling the lac operon. Mutants with a mutation in the lac promoter will typically be Lac , that is, the promoter will no longer function or at least will show decreased expression. However, mutants which cannot make the lactose repressor, the product of the lacI gene, or which make a repressor that cannot bind the inducer, will remain Lac but will constitutively express the products of the operon. Such mutants will grow on the sugar raffinose, which
requires the lactose permease for entry into the cells but is not an inducer of the operon. Constitutive expression of b-galactosidase can also be monitored using the chromogenic compound X-gal (5-bromo-4chloro-3-indolyl-b-d-galactosidase) which is also not an inducer of the operon. However, lacI mutants are also known which lead to repressor binding to lacO, the lactose operation even in the presence of an inducer. These mutants will be phenotypically Lac , and the mutation will be dominant to the wild-type lacI allele. Similarly, most mutations in lacO should diminish or destroy the ability of this site to bind the repressor and lead to constitutive formation of the lac operon enzymes. However, some mutations in lacO lead to enhanced binding and the mutants are Lac . Note that because lacO is a noncoding regulatory region on the DNA, mutations in it will only have an effect on the operon of which they are a part; that is, they will only operate in cis. On the other hand, lacI mutations will function in trans. The ability to make partial diploid strains of E. coli was a very important tool in these Lac mutants. The lac operon, like many others in E. coli, is also positively controlled by the level of cyclic AMP (cAMP) and the cAMP binding protein (catabolite activator protein, CAP), encoded by the crp gene. Mutations in the genes controlling the level of cAMP or the production of CAP will also be phenotypically Lac . However, such mutations will be very pleomorphic, and it would be unusual to refer to them as `Lac mutants.' Interestingly, amino acid residues can be added to the amino terminus of b-galactosidase without important effects on enzyme activity. Therefore lacZ is unusually insensitive to insertion mutations in this region if they maintain the correct reading frame. Because of this, many cloning vectors have been designed to contain a reporter which consists of a multiple cloning site, or polylinker, inserted into this region of the lacZ gene. Essentially all that is required is that the synthetic cloning site does not lead to a frameshift of termination of translation. DNA fragments which are subsequently inserted into such a multiple cloning site will typically introduce such mutations, and
1070
l a c Operon
therefore clones which contain inserts can be readily identified by screening. See also: Constitutive Expression; lac Operon; Lederberg, Joshua; Phenotype
lac Operon J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0738
The lactose or lac operon of Escherichia coli is a cluster of three structural genes encoding proteins involved in lactose metabolism and the sites on the DNA involved in regulation of the operon. The three genes are: (1) lacZ, which encodes the enzyme b-galactosidase (which splits lactose into glucose and galactose); (2) lacY, which encodes lactose permease; and (3) lacA, which encodes a lactose transacetylase. Functional b-galactosidase and lactose permease are required for the utilization of lactose by this bacterium. These proteins are present in the cell in very low amounts when the organism is grown on carbon sources other than lactose. However, the presence of lactose and related compounds leads to the induction of the synthesis of these proteins. Interest in understanding the induction of b-galactosidase by its inducer, lactose, led Jacques Monod and his associates to begin studying the regulation of lactose metabolism in the 1940s. These studies were aided by analogs of lactose that could also be synthesized. Of equal importance, genetic systems (conjugation and transduction) for E. coli were known which enabled genetic analysis of mutants with alterations in lactose metabolism. Throughout the 1950s, Jacques Monod, FrancËois Jacob, and their colleagues performed physiological and genetic experiments on lactose metabolism in E. coli that led to important breakthroughs in our understanding of gene expression and regulation. It was found that some inducers were not substrates of b-galactosidase and some substrates were not inducers. Elegant genetic experiments involving lac mutants led in turn to the discovery of regulatory genes such as lacI, which encoded the lac repressor. These and other experiments led to the operon model of gene expression proposed in 1961. The power of this model was widely appreciated; Jacob and Monod won the Nobel Prize in 1966. The genes in an operon are transcribed into a single, polycistronic messenger RNA (mRNA), in this case from the lac promoter lacP. The regulatory sites that are part of the operon also include the lac operator
lacO. When the lactose repressor binds to lacO, a region immediately upstream of the structural genes of the lac operon, it prevents transcription of the operon. This is an example of negative control. Inducers of the operon bind to the repressor and cause a conformational change that leads to the disassociation of the repressor from the operator. Transcription of the operon then begins. (Although the gene encoding the lactose repressor is not part of the lac operon, it is located next to it on the chromosome.) Later it was discovered that there is another regulatory protein, which participates in positive control of the lac operon. This is the catabolite activator protein (CAP; also called the cAMP receptor protein, CRP), which, when bound to cAMP, itself binds to a region of the lac operon upstream of the promoter and allows RNA polymerase binding. The CAP protein is involved in regulation of many operons as part of a global control system, catabolite repression, which allows the efficient integration of the metabolism of different carbon sources. The E. coli lac operon is of much more than historical importance. Not only has it proved extremely useful as a model for studies of gene regulation, it is also a powerful tool in genetic analysis. For example, the ease of assaying b-galactosidase, both in vitro using colorimetric assays and on plates using chromogenic substrates, has made lacZ an ideal reporter gene in a large variety of experimental situations. In addition, the regulatory system consisting of the lac repressor and lac operator is often incorporated into cloning vectors to provide an easily controlled regulatory system for cloned genes. See also: Catabolite Repression; Cloning Vectors; Induction of Transcription; Jacob, FrancËois; lac Mutants; Monod, Jacques; Operators; Operon; Polycistronic mRNA; Promoters; Regulatory Genes
Lactose J H Miller Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0741
A disaccharide (two sugars joined by an O-glycosidic bond) commonly found in milk. Lactose is termed a b-galactoside because it consists of galactose joined to glucose via a b (1!4) glycosidic linkage. Lactose is cleaved by the enzyme b-galactosidase to yield galactose and glucose. The study of the regulation of b-galactosidase synthesis in bacteria by Jacques Monod
Lac mutants are organisms that contain mutations in some part of the lac operon or its controlling elements. Therefore, they contain some defect in the metabolism of the disaccharide lactose, or in the regulation of this metabolism, when compared with wildtype strains. Lac mutants are of historic interest because they helped to uncover the structure and regulation of the lac operon, the first operon discovered. They are also of interest because the techniques which were developed to screen or select these mutants are still used in the classroom and the laboratory. Wild-type strains of the bacterium Escherichia coli are phenotypically Lac, meaning they have the ability to use lactose as a sole source of carbon. In order to be Lac, E. coli must be able to express a functional lacZ gene, which encodes b-galactosidase, and a functional lacY gene, which encodes the lactose permease. Mutants in which either of these genes have been inactivated are said to be Lac and cannot utilize lactose. Joshua Lederberg and his associates were the first to isolate and map Lac mutants of E. coli, beginning in the 1940s. Lac mutants can be identified by their failure to grow when lactose is the sole carbon source or by the use of various types of indicator plates. Mutations in lacZ or lacY can be differentiated by a variety of techniques. For example, mutants which cannot produce the lactose permease also cannot grow on melibiose under certain conditions. Mutations are also known in the regulatory genes or regions controlling the lac operon. Mutants with a mutation in the lac promoter will typically be Lac , that is, the promoter will no longer function or at least will show decreased expression. However, mutants which cannot make the lactose repressor, the product of the lacI gene, or which make a repressor that cannot bind the inducer, will remain Lac but will constitutively express the products of the operon. Such mutants will grow on the sugar raffinose, which
requires the lactose permease for entry into the cells but is not an inducer of the operon. Constitutive expression of b-galactosidase can also be monitored using the chromogenic compound X-gal (5-bromo-4chloro-3-indolyl-b-d-galactosidase) which is also not an inducer of the operon. However, lacI mutants are also known which lead to repressor binding to lacO, the lactose operation even in the presence of an inducer. These mutants will be phenotypically Lac , and the mutation will be dominant to the wild-type lacI allele. Similarly, most mutations in lacO should diminish or destroy the ability of this site to bind the repressor and lead to constitutive formation of the lac operon enzymes. However, some mutations in lacO lead to enhanced binding and the mutants are Lac . Note that because lacO is a noncoding regulatory region on the DNA, mutations in it will only have an effect on the operon of which they are a part; that is, they will only operate in cis. On the other hand, lacI mutations will function in trans. The ability to make partial diploid strains of E. coli was a very important tool in these Lac mutants. The lac operon, like many others in E. coli, is also positively controlled by the level of cyclic AMP (cAMP) and the cAMP binding protein (catabolite activator protein, CAP), encoded by the crp gene. Mutations in the genes controlling the level of cAMP or the production of CAP will also be phenotypically Lac . However, such mutations will be very pleomorphic, and it would be unusual to refer to them as `Lac mutants.' Interestingly, amino acid residues can be added to the amino terminus of b-galactosidase without important effects on enzyme activity. Therefore lacZ is unusually insensitive to insertion mutations in this region if they maintain the correct reading frame. Because of this, many cloning vectors have been designed to contain a reporter which consists of a multiple cloning site, or polylinker, inserted into this region of the lacZ gene. Essentially all that is required is that the synthetic cloning site does not lead to a frameshift of termination of translation. DNA fragments which are subsequently inserted into such a multiple cloning site will typically introduce such mutations, and
1070
l a c Operon
therefore clones which contain inserts can be readily identified by screening. See also: Constitutive Expression; lac Operon; Lederberg, Joshua; Phenotype
lac Operon J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0738
The lactose or lac operon of Escherichia coli is a cluster of three structural genes encoding proteins involved in lactose metabolism and the sites on the DNA involved in regulation of the operon. The three genes are: (1) lacZ, which encodes the enzyme b-galactosidase (which splits lactose into glucose and galactose); (2) lacY, which encodes lactose permease; and (3) lacA, which encodes a lactose transacetylase. Functional b-galactosidase and lactose permease are required for the utilization of lactose by this bacterium. These proteins are present in the cell in very low amounts when the organism is grown on carbon sources other than lactose. However, the presence of lactose and related compounds leads to the induction of the synthesis of these proteins. Interest in understanding the induction of b-galactosidase by its inducer, lactose, led Jacques Monod and his associates to begin studying the regulation of lactose metabolism in the 1940s. These studies were aided by analogs of lactose that could also be synthesized. Of equal importance, genetic systems (conjugation and transduction) for E. coli were known which enabled genetic analysis of mutants with alterations in lactose metabolism. Throughout the 1950s, Jacques Monod, FrancËois Jacob, and their colleagues performed physiological and genetic experiments on lactose metabolism in E. coli that led to important breakthroughs in our understanding of gene expression and regulation. It was found that some inducers were not substrates of b-galactosidase and some substrates were not inducers. Elegant genetic experiments involving lac mutants led in turn to the discovery of regulatory genes such as lacI, which encoded the lac repressor. These and other experiments led to the operon model of gene expression proposed in 1961. The power of this model was widely appreciated; Jacob and Monod won the Nobel Prize in 1966. The genes in an operon are transcribed into a single, polycistronic messenger RNA (mRNA), in this case from the lac promoter lacP. The regulatory sites that are part of the operon also include the lac operator
lacO. When the lactose repressor binds to lacO, a region immediately upstream of the structural genes of the lac operon, it prevents transcription of the operon. This is an example of negative control. Inducers of the operon bind to the repressor and cause a conformational change that leads to the disassociation of the repressor from the operator. Transcription of the operon then begins. (Although the gene encoding the lactose repressor is not part of the lac operon, it is located next to it on the chromosome.) Later it was discovered that there is another regulatory protein, which participates in positive control of the lac operon. This is the catabolite activator protein (CAP; also called the cAMP receptor protein, CRP), which, when bound to cAMP, itself binds to a region of the lac operon upstream of the promoter and allows RNA polymerase binding. The CAP protein is involved in regulation of many operons as part of a global control system, catabolite repression, which allows the efficient integration of the metabolism of different carbon sources. The E. coli lac operon is of much more than historical importance. Not only has it proved extremely useful as a model for studies of gene regulation, it is also a powerful tool in genetic analysis. For example, the ease of assaying b-galactosidase, both in vitro using colorimetric assays and on plates using chromogenic substrates, has made lacZ an ideal reporter gene in a large variety of experimental situations. In addition, the regulatory system consisting of the lac repressor and lac operator is often incorporated into cloning vectors to provide an easily controlled regulatory system for cloned genes. See also: Catabolite Repression; Cloning Vectors; Induction of Transcription; Jacob, FrancËois; lac Mutants; Monod, Jacques; Operators; Operon; Polycistronic mRNA; Promoters; Regulatory Genes
Lactose J H Miller Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0741
A disaccharide (two sugars joined by an O-glycosidic bond) commonly found in milk. Lactose is termed a b-galactoside because it consists of galactose joined to glucose via a b (1!4) glycosidic linkage. Lactose is cleaved by the enzyme b-galactosidase to yield galactose and glucose. The study of the regulation of b-galactosidase synthesis in bacteria by Jacques Monod