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The Lac repressor binds to the lac operatsr in vitro

Posted by star on 2018-11-05 22:22:40
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    An important step in proving the principal hypothesis of the operon model was the isolation of the Lac repressor and the demonstration of its expected properties. Walter Gilbert and Benno Mullier-Hill succeeded in isolating the Lac repressor from E. coli extracts.They fractionated proteins by standard techniques and then assayed individual fractions for their ability to bind [14C]IPTG (one of the gratuitous inducers). Binding was detected by equilibrium dialysis.
    The Lac repressor is a homotetramer with a molecular mass of 154 kDa. Each subunit, which is made of 360 amino acids, can bind one molecule of IPTG. Crude cell extracts bind about 20 to 40 molecules of IPTG per cell, so there are roughly 5 to 10 repressor molecules per cell. Support for the idea that the IPTG-binding protein is the lac repressor comes from the observation IPTG-binding protein is absent in extracts of ldcl- mutants. Still stronger support comes from the observation that lacI mustants, which introduce amino acid substitutions in lac repressor, alter the repressor's affinity for IPTG.
    Because the number of repressor molecules is extremely small, these molecules must be translated from no more than one or two repressor mRNA molecules transcribed per generation time. The number of mRNA molecules is so small that either repressor synthesis itself is regulated or the mRNA is transcribed from a weak promoter. Both mechanisms have been observed for regulation of repressor synthesis in other operons, but for the Lac repressor the second explanation is correct, that is, repressor mRNA is transcribed constitutively from a weak promoter. The reason for the small number of repressor molecules is made clear from the properties of several mutants in which the weak lacI promoter is converted to a strong promoter. These mutants are noninducible because it is not possible to fill a cell with enough inducer to overcome repression.......

Reveal a new inflammation control mechanism

Posted by star on 2018-11-05 17:50:40
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    In the event of infection or tissue damage, an inflammatory immune response attacks the infection and repairs the damaged tissue. However, sometimes excessive inflammation has the opposite effect: in a process called immunopathology. This will increase tissue damage. In a new study, researchers from CNIC have discovered a new mechanism of inflammation control that demonstrates how to control the tissue damage caused by inflammatory immune response. The results of the study were published in Science on October 19, 2018 , entitled "DNGR-1 in dendritic cells limits tissue damage by inhibiting neutrophil recruitment."
    The first immune cells that reach the site of infection or inflammation are neutrophils, and the task of these cells is to eliminate the root cause of this problem. However, neutrophils are very destructive, not only acting on infectious pathogens, but also destroying damaged tissues. This tissue damage caused by our own defense system is called an immunopathological response. This new study confirms that neutrophil infiltration into tissues is controlled by dendritic cells. It is well known that these dendritic cells play an important role in guiding the specific response of T lymphocytes. This new study suggests that dendritic cells regulate neutrophil infiltration to help avoid excessive tissue damage.
    Dendritic cells attract neutrophils into inflammatory focus by releasing factors such as chemokine Mip-2. At the same time, these dendritic cells also express the surface receptor DNGR-1. The cell surface molecules detect tissue damage by recognizing cellular components that are only accessible when the cells are damaged or "ruptured". When DNGR-1 detects damaged tissue, it reduces the ability of dendritic cells to produce Mip-2, thereby limiting neutrophil infiltration into damaged organs. This mechanism prevents the expansion of tissue damage that may be life-threatening.


The inducer of the lactose operon

Posted by star on 2018-11-05 00:06:08
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    Two related problems became evident as the operon model was tested. First, inducers must enter a cell if they are to bind to repressor molecules, yet lactose transport requires permease, and permease synthesis requires induction. Thus, we must explain how the inducer gets into a cell in the first place. Second, the isolated Lac repressor does not bind lactose (4-O-β-D-galactopyranosyl-D-glucose) but does bind a lactose isomer called allolactose (6-O-β-D-galactopyra-nosyl-D-glucose). Remarkably, β-galactosidase, the enzyme that catalyzes lactose hydrolysis, also converts a small proportion of lactose to allolactose. Therefore, induction of the synthesis of β-galactosidase by lactose requires that β-galactosidase be present.


    Both problems are solved in the same way in the uninduced state, a small amount of lac mRNA is synthesized (roughly one mRNA molecule per cell per generation). This synthesis, called basal synthesis, occurs because the binding of repressor to the operator is never infinitely strong. Thus, even though the repressor binds very strongly to the operator, it occasionally comes off and an RNA polymerase molecule can initiate transcription during the instant that the operator is free.
    We can now describe in molecular terms the sequence of events following addition of a small amount of lactose to a growing Lac+ culture. Consider bacteria growing in a medium in which the carbon source is glycerol. Each bacterium contains one or two molecules of β-galactosidase and of lactose permease. Lactose is then added. The few permease moIecules transport a few lactose molecules into the cell and the few β-galactosidase molecules convert some of these lactose molecules into allolactose. An allolactose......

The regulation of the lactose system

Posted by star on 2018-11-01 22:48:27
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    Monod and Jacob proposed the operon model explain how the lac system is regulated. The term operon refers to two or more contiguous genes and the genetic elements that regulate their transcription in a coordinate fashion. Promoters had not yet been discovered when Monod and Jacob proposed the operon model but were readily incorporated into the operon model after their discovery. Lac operon mode shows a revised version of the original lac operon model that includes the lac promoter. The five major features of the model are:
    1. The products of the lacZ, lacY and lacA genes are encoded in a single polycistronic lac mRNA molecule.
    2. The promoter for this mRNA molecule is immediately adjacent to the lacO region. Promoter mutations (P-) that are completely incapable of making β-galactosidase, permease, and transacetylase have been isolated. The promoter is located between lacI and lacO.
    3. The operator is a sequence of bases (in the DNA) to which the repressor protein binds.
    4. When the repressor protein is bound to the operator; transcription of lac mRNA cannot take place.
    5. Inducers stimulate lac mRNA synthesis by binding to the repressor. This binding alters the repressor's conformation so it cannot bind to the operator. Thus, in the presence of an inducer the operator is unoccupied and the promoter is available for initiation of mRNA synthesis. This state is called derepression.
    This simple model explains many of the features of the lac system and of other negatively regulated genetic systems. However we will see in a later section that this explanation is incomplete as the lac operon is also subject to positive regulation.



lac mRNA

Posted by star on 2018-10-31 23:13:09
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    Two French investigators, Jacques Monod and Francois Jacob, some-times working together and sometimes with other investigators, performed a series of genetic and biochemical experiments in the late 1950s that helped to elucidate the mechanism that regulates the lac system, They began by isolating constitutive E. coli mutants that make lac mRNA in the presence as well as absence of an inducer. Then they constructed a variety of partial diploid cells containing constitutive mutants and observed the cell's ability to synthesize β-galactosidase.

    These mutations appeared to be of two types, termed lacI and lacOc. The lacI- mutations behave like typical minus mutations in most genes and are recessive (entries 3, 4). Because lac mRNA synthesis is off in a lacl+ cell and on in a lacI- mutant, the lacI gene is apparently a regulatory gene that codes for a product that acts as an inhibitor to keep the lac structural genes turned off. A lacI-mutant lacks the inhibitor and thus is constitutive. A lacl+/lacI- partial diploid has one good copy of the lacI- gene product, so the system is inhibited. Monod and Jacob called the lacI- gene product the Lac repressor. Their original genetic experiments did not indicate whether the Lac repressor is a protein or an RNA molecule.

    This question was answered when an E. coli mutant with a polypeptide chain termination codon inside lacI was isolated and found to synthesize β-galactosidase constitutively. The most likely explanation for the constitutive lactose system is that the mutant strain synthesizes a truncated repressor protein that cannot block transcription of the lac genes. This conclusion was confirmed when the Lac repressor was purified and chara......

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