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Constitutive mutation in operator gene

Constitutive mutation in operator gene


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If a constitutive mutation happens in the operator of an inducible operon, does that mean that repressors won't be able to bind them ? Or does it mean that even if repressors are bound, they will not have any effect on the gene ?

I am specifically talking about lac operon.


For the lac operon there are two possibilities for constitutive expression mutations:

  1. The operator is never closed.

Reason: Mutation of the repressor, so its not present, doesn't bind or binds only with very low affinity for the operon.

  1. The repressor can not bind.

Reason: The binding site for the repressor is mutated.

See this Website or this Website for more information.


Operon

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. [1] The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon. [2]

Originally, operons were thought to exist solely in prokaryotes (which includes organelles like plastids that are derived from bacteria), but since the discovery of the first operons in eukaryotes in the early 1990s, [3] [4] more evidence has arisen to suggest they are more common than previously assumed. [5] In general, expression of prokaryotic operons leads to the generation of polycistronic mRNAs, while eukaryotic operons lead to monocistronic mRNAs.

Operons are also found in viruses such as bacteriophages. [6] [7] For example, T7 phages have two operons. The first operon codes for various products, including a special T7 RNA polymerase which can bind to and transcribe the second operon. The second operon includes a lysis gene meant to cause the host cell to burst. [8]


Single mutants of the lac operon

The lac operon and its regulators were first characterized by studying mutants of E. coli that exhibited various abnormalities in lactose metabolism. Some mutants expressed the lac operon genes constitutively, meaning the operon was expressed whether or not lactose was present in the medium. Such mutant are called constitutive mutants.

The operator locus (lacO) - One example is O c, in which a mutation in an operator sequence and reduces or precludes the repressor (the lacI gene product) from recognizing and binding to the operator sequence. Thus, in O c mutants, lacZ, lacY, and lacA are expressed whether or not lactose is present.

The lacI locus &ndash One type of mutant allele of lacI (callled I -) prevents either the production of a repressor polypeptide or produces a polypeptide that cannot bind to the operator sequence. This is also a constitutive expresser of the lac operon because absence of repressor binding permits transcription.

Another type of mutant of lacI called I s prevents the repressor polypeptide from binding lactose, and thus will bind to the operator and be non-inducible.. This mutant constitutively represses the lac operon whether lactose is present or not. The lac operon is not expressed and this mutant is called a super-repressor.


Function of Regulator Gene | Genetics

In this article we will discuss about the function of regulator gene.

The genetic basis for induction and repression was studied for several years by Jacob and Monod at the Pasteur Institute in Paris. They investigated regulation of the activities of genes which control fermentation of lactose through synthesis of the enzyme β-galactosidase in E. coli. They were awarded Nobel Prize in 1965.

If wild type E. coli cells are grown on a medium containing glucose, the cells are not able to utilise lactose and contain very small quantities of the enzyme β-galactosidase. But if wild type E. coli are grown on a medium devoid of glucose, but containing lactose as the only carbon source, within two minutes they start synthesizing β-galactosidase.

The synthesis of enzyme continues until very large amounts (about 3000 molecules per cell) have been produced. It was found that along with β-galactosidase, lactose induces the synthesis of two other enzymes viz. β-galactoside permease, which facilitates entry of lactose into the cells and β-galactoside transacetylase, whose function is obscure.

The three collectively are known as lac enzymes. Jacob and Monod studied gene regulation by isolating lactose mutants of E. coli which had one defect or the other in this regulation.

The mutants revealed following different types of genes performing different functions in regulation:

(a) There are mutants which on growing on lactose medium, do not have one of the three enzymes synthesised on induction. Mapping techniques have shown that they have defects in three adjacent genes, each of which directs the synthesis of one of the enzymes. These are called structural genes and were shown by Lederberg and his colleagues to be arranged continuously on the chromosome in the order β-galactosidase (denoted z gene), permease (y) and transacetylase (a).

(b) Constitutive Mutants: Enzymes may be constitutive or induced. Constitutive enzymes are those made in constant amounts in a cell, without regard to the metabolic state of the cell. Induced enzymes are made when required in response to the presence of their substrates in a cell.

Constitutive mutants of E. coli studied by Jacob and Monod are those that synthesise the three enzymes regardless of the presence or absence of the inducer. The gene showing this defect was called the regulator gene (denoted by i) and was found by mapping techniques to lie before the z gene.

(c) E. coli cells which were diploid as they had one complete chromosome and a second chromosome fragment homologous with a portion of the first chromosome. Such a bacterial cell is partially diploid for some genes and is called meroploid. Mutants with two chromosomes in a cell were analysed as follows: one chromosome had an active i but a defective z gene (i + z – ) the other chromosome having active z but defective i gene (i – z + ).

Such mutants produce β-galactosidase only in presence of inducer. It means that the active regulator gene (i + ) on one chromosome can regulate the active structural gene (z + ) on the other chromosome. Obviously, the regulator gene must be controlling the synthesis of an intermediary molecule which diffuses through the cytoplasm.

Some other experiments showed that the regulator gene codes for the amino acid sequence of a specific protein called repressor. The repressor molecule diffuses from the ribosomes where it is formed and becomes physically bound to a specific site on DNA near the structural gene.

(d) Further understanding of the repressor molecule came from mutants which were constitutive even though they had an active regulator gene. Such mutants failed to respond to the repressor because of a defect in a small specific region of the chromosome to which the repressor becomes bound. This was called the operator (denoted O) situated near the beginning of the β-galactoside structural gene (z).

The existence of operators was first revealed by genetic analysis. A mutation in the operator can make it inactive, preventing the binding of the repressor. When this happens, then constitutive enzyme synthesis occurs on the z, y and a genes. These mutants are therefore called operator constitutive O c mutants.

The operator constitutive mutants can be distinguished from mutations in repressor genes by measuring enzyme synthesis in partially diploid cells for certain chromosomal regions.

If such a partially diploid cell contains one mutant and one functional repressor gene, repression occurs because repressor molecules produced by one functional locus can bind to both operators. But if there is one non-functional operator locus, the cells would always be constitutive.

From genetic studies in mutants combined with biochemical evidence, Jacob and Monod derived the following conclusions: the lac operon regulates the metabolism of lactose. When E. coli cells are grown on a medium containing lactose, the lac operon becomes functional and synthesizes enzymes required for the transport and breakdown of lactose.

The lac operon does not function when glucose is present or when lactose is absent from the medium. The lac operon contains a promoter (p), an operator (o), and three structural genes (z, y and a). It also has a transcription terminator gene (t) which gives the chain termination signal during mRNA synthesis.

The regulator gene directs the formation of a repressor protein. This protein has affinity for the sequence of nucleotides of the operator and can bind to the operator. When the repressor is bound to the operator it prevents movement of RNA polymerase towards the three structural genes no mRNA is synthesised, and therefore the three proteins are not formed.

When inducer (lactose) is present, its molecules can bind to another active site of the repressor protein. This binding changes the three dimensional conformation of the protein, so that it loses its affinity for the operator. The operator is made free, mRNA is transcribed by the structural genes and all the three enzymes are synthesised (Fig. 16.1).

Enzyme Repression:

Jacob and Monad also postulated repression of enzyme synthesis. For example, if histidine is added to the culture medium in which E. coli cells are growing, the enzymes leading to the formation of histidine become repressed, and histidine is not synthesised. The process is called feedback inhibition or end-product repression.

By itself the repressor molecule is inactive. But when a co-repressor binds with it, the repressor-co-repressor complex binds with the operator gene that is specific for the structural genes of this operon, and prevents transcription. Thus there are two types of repressor molecules, one which binds with the inducer and promotes synthesis of enzymes the other binds the co-repressor resulting in end-product repression.


Constitutive mutants are those strains in which a protein is continuously produced, which in wild is inducible. For example, the constitutive mutant strain with lac operon mutation is responsible for the transcription of the lac genes, even in the absence of lactose in the medium.

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Details of Structural Genes | Genetics

In this article we will discuss about the details of structural genes.

Structural Genes:

They control amino acid sequence of a protein by producing mRNA. There are as many structural genes as there are proteins which are regulated. Either a single mRNA is transcribed from each structural gene or, all the structural genes of an operon form a single polycistronic mRNA.

The Operator:

It is the binding site for the repressor and controls the structural genes. Itself it is under the negative control of the repressor protein. The operator determines whether or not structural genes are to be repressed by the repressor produced by regulatory gene. If there is a mutation in the operator region rendering it functionless, the repressor is not able to bind and structural genes are transcribed. Mutants of the operator locus are called operator-constitutive mutants (O c ).

Gilbert and his colleagues isolated the operator region by breaking DNA of lac region into fragments 1,000 base pairs long. A property of the operator region is that when it is complexed to the repressor it is protected from digestion by DNAase. The operator region has double helical DNA about 27 base pairs long.

The Promoter:

It consists of a small segment of DNA, less than 100 nucleotides in length which lies between the regulatory gene and the operator gene. It has a binding site which is recognised by the enzyme DNA directed RNA polymerase and which gives the initiation signal for transcription of mRNA.

RNA polymerase moves along the operator locus and over to the structural genes which transcribe mRNA. Besides the lac operon, in other operons, the promoter region has a second binding site for a specific protein called the cyclic AMP receptor protein.

The Lac Operon:

The structural genes z, y and a, together with the operator o constitute the lactose operon. The operon provides a mechanism for the coordinate expression of structural genes (under the control of the operator and the regulator) resulting in the induction of enzymes due to a single inducer.

The operator locus controls the transcription of the entire group of coordinately induced genes so that a single large polycistronic messenger RNA is formed. The enzyme RNA polymerase binds to the promoter and all the genes in the operon are transcribed in a sequence. When repressor produced by the regulator is bound to the operator, transcription is not initiated and expression of all the genes in the operon is inhibited simultaneously.

Another feature of the operon is polarity. The genes z, y and a synthesise equal quantities of the three enzymes β-galactosidase, permease and acetylase and in the same order as their respective genes are located in DNA. Thus β-galactosidase (product of z gene) is synthesised first, followed by permease (y gene product), and last of all acetylase (produced by a gene).

This is the effect of polarity. If there is mutation in z gene, there is no transcription of all three structural genes. If the mutation is in the y gene, then both y and a genes are inhibited, whereas the z gene synthesizes β-galactosidase. These are called polar mutations.

Generally speaking we can say that an operon is a group of functionally related structural genes which can be turned on or off coordinately under the control of the same regulatory gene. The clustering of genes for various enzymes of a single metabolic pathway is probably necessary to facilitate operon function.

The Regulator Gene:

It determines whether structural genes will be transcribed or not. It codes for the amino acid sequence of a specific repressor protein. The repressor molecule diffuses from the ribosomes where it is formed and becomes bound to the operator. Due to the diffusible nature of its product repressor, the regulator gene does not always lie adjacent to the structural genes it regulates.

When the regulator gene undergoes mutation, it can no longer inhibit the transcription of the structural genes. The genes z, y and a then synthesise the three enzymes whether the inducer is present or not. For a long time the chemical nature of the repressor molecules postulated by Jacob and Monod could not be identified.

In 1967, W. Gilbert and Muller-Hill succeeded in isolating the lac repressor. They produced mutant E. coli cells which contained almost ten times the amount of lac repressor present in normal cells. The repressor protein has now been crystallized. It has a molecular weight of about 150,000 with a high affinity for its specific locus in E. coli. Besides the lac repressor, the galactose and tryptophan repressor have also been isolated in a pure form.

The lac repressor is a tetramer consisting of four units of a protein coded by the regulator gene, each of molecular weight about 37000. An E. coli cell contains about 10 such repressor molecules. There are almost 347 amino acid residues in a repressor. About 50 amino acids in the lac repressor bind to a sequence of about 12 base pairs in the operator region. This binding of the repressor prevents transcription of mRNA by RNA polymerase.

When lactose is present in the medium its uptake into the E. coli cell is followed by trans- glycosylation, a slight molecular rearrangement to form allolactose (Fig. 16.2). The synthesis of allolactose is catalysed by the few β-galactosidase molecules that are present prior to induction. Allolactose binds to the lac repressor to form an inducer-repressor complex.

When the inducer becomes bound to the repressor, the binding of the repressor to the operator region is released due to a change in the 3-dimensional conformation of the repressor protein called allosteric effect. The unbound operator then permits RNA polymerase to transcribe. In this case allolactose is the effector molecule which prevents the regulatory protein from binding to the operator.


Constitutive mutation in operator gene - Biology

Example: Two white-flowered plants cross to produce purple flowers , although purple is dominant.
Each contains a mutation in a different gene, encoding a different enzyme needed to make the purple pigment.

Complementation analysis is easiest to do in bacteria, fungi, or C. elegans, where many mutants of a given phenotype can be obtained.

If we isolate a large number of strains with the same defective phenotype,we can cross them in all combinations, and figure out the number of complementation groups. Any two defective strains that FAIL to complement are in the same complementation group. Usually each complementation group represents one of the essential enzymes in the pathway.

Problem 1: Figure out how many complementation groups there are in these examples.

The concept of complementation is extremely important in molecular biology. For example, the sickle-cell mouse line could only be created because two strains with different defects (lack of mouse or human globin genes) could be mated to complement each other's defects. The fact that genes from different species can complement each other was one of the most significant conceptual advances in molecular biology. Complementation is now used routinely to answer more subtle questions of how genes are regulated. You absolutely need to understand complementation to understand molecular biology.

Bacterial complementation.
Bacterial genetic systems can show complementation in two important ways--each manipulating a natural process of bacterial genetics. These two processes have since been modified in biotechnology to provide most of the essential tools of gene cloning.

1. Specialized Transduction. A lysogenic bacteriophage can excise itself so as to carry a piece of host DNA by mistake. The phage will now carry a second copy of an allele (or linked alleles) into a host cell. The new bacterium is a partial diploid for the allele(s).

In biotechnology, a phage chromosome can have a piece of foreign DNA ligated into it in the test tube. Then the phage DNA is packaged into phage, and it can infect a new host where it either (1) produces many copies of the host gene or (2) lysogenizes the host, to express the cloned DNA.

2. F' plasmid. The F plasmid can recombine itself into the host chromosome, then recombine itself out again with some host DNA by mistake. When it enters the next host cell, it carries a second copy of several genes again, a partial diploid is created.

In biotechnology, a plasmid can have a piece of foreign DNA ligated into it in the test tube then the plasmid is transformed into E. coli. Then the plasmid makes many copies, including the cloned gene.

Suppressor mutation analysis.
A variation on complementation is suppressor mutations. A suppressor mutation corrects a defect in a different gene locus. A mutant version of gene A makes an altered gene product, which corrects the phenotype of a defective mutation in gene B.

Organization of Genes
In bacteria, a number of gene ORFs can be organized into an operon . All the gene sequences in a given operon are transcribed on a single mRNA, starting at one promoter . An example of an operon is shown, tuf-s10 , from Borrelia burgdorferi, which causes Lyme Disease:

  • elongation factor (tuf)
  • ribosomal proteins S10 (rpsJ)
  • L3 (rplC)
  • L4 (rplD)
  • L23 (rplW)
  • L2 (rplB)
  • S19 (rpsS)
  • L22 (rplV)
  • S3 (rpsC)

Human Growth Hormone Receptor

For other interesting operons, try searching GenBank, the international repository for all known DNA sequences. (Funded by the U.S. government--your tax dollars at work.)

  • DNA sequence--inversion or deletion
  • Transcription, sigma factor
  • Transcription, promoter sequence, repressor proteins
  • Translational repressor
  • Posttranslational modification

The Lac Operon
The Lac operon is the classic model for activation and repression of transcription. Concepts of analysis based on the Lac operon can be applied to other systems including animals and plants.

The following explanation of the Lac operon is modified from MIT Lac Operon.
Jacob and Monod were the first scientists to elucidate a transcriptionally regulated system. They worked on the lactose metabolism system in E. coli. When the bacterium is in an environment that contains lactose:

It should turn on the enzymes that are required for lactose degradation. These enzymes are: beta-galactosidase: This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. It is coded for by the gene LacZ. Lactose Permease: This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY. Thiogalactoside transacetylase: The function of this enzyme is not known. It is coded for by the gene LacA. The sequences encoding these enzymes are located sequentially on the E. coli genome. They are preceded by the LacI region which regulates expression of the lactose metabolic genes. You might expect that the cell would want to turn these genes on when there is lactose around and off when lactose is absent. But the story is more complicated than that. For instance, the permease gene always needs to be expressed at a low level, in order for any lactose to get into the cell. So a certain low level of expression is constitutive --that is, occurs all the time, even if "repressed." Most bacterial operons are partly or totally constitutive. LacI expression, for example, is totally constitutive its promoter is always "turned on," for a very low level of expression, just enough to make a few repressor molecules.

A bacterium's prime source of food is glucose, since it does not have to be modified to enter the repiratory pathway. So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favour of glucose metabolism. There are regulatory sites upstream of the Lac genes that respond to glucose concentration.

  • Lactose induces transcription by pulling the LacI repressor off.
  • Glucose prevents transcription by pulling the CAP activator off.

When lactose is present, it acts as an inducer of the operon. It enters the cell, rearranges slightly to form allolactose, then binds to the Lac repressor. A conformational change causes the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA, and RNA can be made from the three structural genes . The mRNA will be translated to the proteins which transport and metabolize lactose.

When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made.

Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription the promoter doesn't have to wait for the holoenzyme to bind. Catabolite Repression, with an Activator Protein
When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. But when glucose levels drop, ATP phosphates are released until at last forming cAMP:

ATP --> ADP + Pi --> AMP + Pi --> cAMP

cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression , a misnomer since it involves an activator protein , but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons.

This image shows a "close-up" view of CAP regulation:

Corepressor control
Other operons are controlled by their products, rather than their substrates for example, expression of biosynthetic enzymes to build amino acids. This is called feedback inhibition. In the Trp operon, for tryptophan biosynthesis, transcription of mRNA for five enzymes is prevented by binding of the Trp corepressor in the presence of tryptophan. When tryptophan levels fall, Trp comes off of the corepressor, and the corepressor comes off of the promoter/operator site. Transcription now occurs, so that the cell has enzymes to make more tryptophan.
Analysis of operon control
What experiments do we perform to figure out how operons are regulated?
We use partial diploid strains created by F' or specialized transduction. In either case, we test what happens when a strain is diploid for regulatory elements.

Regulatory mutants can have various kinds of mutant phenotypes. For example:

p- Promoter fails to bind RNA polymerase. No transcription occurs.
lacI- Repressor fails to bind promoter/operator. Transcription occurs constitutively
(in the presence or absence of lactose)
o-c Operator fails to bind repressor. Transcription is constitutive.
lacZ- Structural gene is defective. No enzyme is made.

What will happen? What kinds of complementation can occur?Does is matter if the two mutant alleles are adjacent on the same chromosome ( cis ) or separated ( trans )?


"Wild type" Mutant
LacZ+ Makes B-gal enzyme LacZ- Make NO enzyme
LacI+ Makes Repressor LacI- Makes NO repressor
Transcription can be constitutive
IF PROMOTER IS FUNCTIONAL
p + RNA Pol binds promoter p - RNA Pol does NOT bind promoter
No Transcription
o + Operator binds repressor o - c Operator does NOT bind repressor
Transcription can be constitutive
IF PROMOTER IS FUNCTIONAL

Problem 2. Predict whether the following diploids produce B-galactosidase, in the presence of lactose in the absence of lactose. Explain why. Explain in each case whether it matters if the two mutant alleles are located in cis or in trans.

p + lacZ - lacI +
----------------- ----------
p + lacZ + lacI -

p + lacZ - lacI -
----------------- ----------
p - lacZ + lacI -

p + lacZ - lacI +
----------------- ----------
p + lacZ + lacI -

p + o-c lacZ + lacI + (A constitutive operator NEVER binds repressor,
-------------------------- -------- with or without lactose.)
p + o + lacZ - lacI +

Problem 3. Explain two different genetic processes in bacteria that can create a "partial diploid" for a small part of the genome. Explain why these processes are useful for bacterial genetic analysis.

Problem 4. State whether B-galactosidase is expressed by each lac operon diploid, (1) and (2), and briefly state why (one sentence). Complete possible genotypes for (3) and (4).


Lactose
Absent
Lactose
Present
LacI- P+ O-c LacZ+
LacI+ P+ O+ LacZ-
LacI+ P- O-c LacZ+
LacI+ P+ O+ LacZ-
LacI- P+ O+ ___
____ ___ __ LacZ-
- +
__ P- O-c LacZ+
__ P+ __ ___
+ +

Quiz on Lac Operon -- Highly Recommended

Molecular Structure of Promoters
Promoters are defined by sequences of base pairs upstream of the transcription start site. The RNA polymerase tends to recognize promoter sequences in which most of the base pairs match the promoter consensus sequence. The consensus sequence is a composite defined by the most common base to occur at each position. Base-substitution mutations can decrease or increase the efficiency of the promoter.

Bacterial consensus promoters include two regions of six base pairs each, at -10 and -35 bases upstream. However, no two promoters are exactly alike, and no promoter exactly matches the consensus sequence. Additional sites for environmental regulators can be found as far as -50 to -300 bases upstream.

  • When bacteria use up their carbon sources, they express RpoS, the starvation sigma factor . (Review, what is a sigma factor?)
  • RpoS joins RNA polymerase to initiate transcription of different environmental stress genes--genes protecting against all the different stresses that the bacteria might encounter before they enter a new human intestine. This phenomenon is known as cross-protection.
  • The stress genes can be used for conditions as unrelated as acid or base resistance.
  • The stress genes activated may or may not be part of multi-gene operons. They may face in opposite directions, from many different promoters, at all different loci around the genomic map.
  • Arsenic Resistance Operons. How do bacteria resist arsenic? An environmental response regulon is turned on by arsenic. The molecular basis is related to how cancer cells develop resistance to anti-cancer drugs.
  • Environmental regulation in Yersinia pestis (bubonic plague bacteria). Several complex regulons of genes respond to specific environmental factors, particularly iron and temperature. At low temperature, the presence of iron tells the bacterium, "I am in blood that has been swallowed by a flea." The bacterium expresses proteins that upset the flea's digestion, forcing it to regurgitate the bacteria into the blood of its next victim. (Susan Straley & Robert Perry, Trends in Microbiol., 1995)
  • E. coli virulence regulator. How do virulent E. coli strains kill children? A regulator protein binds to an operon encoding "pilins," for E. coli to make pili which attach to the intestinal epithelium.
  • Tuberculosis model gene expression. How are genes regulated in a tuberculosis-related pathogen?

    M. Donnenberg, U. Maryland
  • Virulence regulator in "Flesh-eating bacteria." A gene activator protein in Staphylococcus aureus turns on the virulence regulon that makes the flesh-eating toxins. This activator can be used as a vaccine against S. aureus.
  • DNA Microarrays. We can now put most of the protein-encoding genes onto a microarray chip, using technology based on the DNA silicon chip industry. The chip can be used to hybridize to cellular RNA, and measure the expression rates of a large number of genes in a cell.

The F-factor and two lac operons in a single cell &ndash partial diploid in E.coli

More can be learned about the regulation of the lac operon when two different copies are present in one cell. This can be accomplished by using the F-factor to carry one copy, while the other is on the genomic E. coli chromosome. This results in a partial diploid in E. coli.

The F-factor is an episome that is capable of being either a free plasmid or integrated into the host bacterial chromosome. This switching is accomplished by IS elements where unequal crossing over can recombine the F-factor and adjacent DNA sequences (genes) in and out of the host chromosome. Researchers have used this genetic tool to create partial diploids (merozygotes) that allow them to test the regulation with different combinations of different mutations in one cell. For example, the F-factor copy may have a I S mutation while the genomic copy might have an O C mutation. How would this cell respond to the presence/absence of lactose (or glucose)? This partial diploid can be used to determine that I S is dominant to I + , which in turn is dominant to I - . It can also be used to show the O C mutation only acts in cis- while the lacI mutation can act in trans- .


Caenorhabditis elegans: Molecular Genetics and Development

Vida Praitis , Morris F. Maduro , in Methods in Cell Biology , 2011

G Marking Extrachromosomal Arrays to Probe Gene Regulation

The interaction of the E. coli LacI protein with lacO lactose operator sequences was exploited as a method for marking chromosomes in yeast ( Belmont and Straight, 1998 ) and has been used as a marker for transgenes in C. elegans as well ( Gonzalez-Serricchio and Sternberg, 2006 ). Use of the LacI/LacO systems has also been used to label extrachromosomal arrays to study gene regulation ( Fig. 3A ). In such experiments, a GFP-tagged endogenous transcription factor is expressed in the presence of an extrachromosomal array that carries a promoter that contains its target cis-regulatory sites. The factor will interact with the many copies of the target promoter in the array, producing a subnuclear spot. LacI tagged with a different marker can label lacO sequences in the same target array, allowing an independent means by which to verify interaction of the GFP-tagged factor with the array ( Carmi et al., 1998 ). Researchers have also used the GFP::LacI/LacO system to demonstrate that transgenes move to different locations in the nucleus depending on whether they are active or inactive in a given cell or tissue ( Meister et al., 2010 ).

Fig. 3 . Examples of types of transgenes and their expression patterns. (A) Expression of a chromosomally-integrated med-1::GFP::MED-1 translational reporter in the early embryo, showing nuclear GFP expression in the daughters of the blastomeres MS and E ( Maduro et al., 2002 ). Due to the presence of a separate extrachromosomal array carrying a transcriptional lacZ reporter for the MED-1 target gene end-3, the GFP::MED-1 localizes to subnuclear spots representing the extrachromosomal array (arrowheads) in each nucleus. (B) Expression of a translational fusion of the adherens junction marker ajm-1 in mid-embryogenesis. GFP becomes localized to adherens junctions, giving an outline of epidermal cells ( Koppen et al., 2001 ). (C) DIC image of a late embryo, just prior to hatching, with the pharynx and intestine indicated. (D) Expression of an elt-2::NLS::YFP::lacZ reporter transgene in the same embryo as in (C) localized to intestinal nuclei (and excluded from nucleoli). (E) A C. elegans adult hermaphrodite showing expression of an unc-119::mCherry transcriptional reporter throughout the nervous system (including the nerve ring, neurons around the vulva, and the ventral nerve cord indicated by arrowheads) and in head muscles (Maduro and Pilgrim, 1995). The head muscle expression has been overexposed. Anterior is to the left. A C. elegans embryo is approximately 50 μm long, while adults are approximately 1mm long. (See color plate.)


Operator-constitutive mutations of the Escherichia coli metF gene.

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nucleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.



Comments:

  1. Dalabar

    Absolutely with you it agree. It seems to me it is very good idea. Completely with you I will agree.

  2. Pontus

    This very good thought has to be purposely

  3. Jutaxe

    SUPER everything, GENERALLY COOUTOO, if it were really so

  4. Gardazahn

    I must tell you.

  5. Nikolas

    Babies the highest grade !!!



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