Question

18. When attempting a targeted gene knockout using mouse embryonic stem cells, there are three possible outcomes: targeted knockout, ectopic insertion, and no insertion. What procedures can be used to select for cells that only have the targeted gene knockout? (9 pts)

19. Describe why Jacob and Monod used IPTG as a synthetic inducer during their experiments investigating the genetic control of the lac operon. (6 pts)

20. Describe the function of the CAP-cAMP system in bacteria. Why does it regulate several operons related to metabolism? (7 pts)

21. Many DNA-binding proteins that affect gene expression are allosteric. What is allostery and why is it such a common mechanism of gene regulation? (7 pts)

Answer 1

PROCEDURE -

neon= Electroporation 707 = Gene of interest 000 neo Homologous recombination Selection with resistance marker = =neo

The procedure for making mixed-genotype blastocyst.

Foster mother Chimera mouse Normal mouse Chimera mouse Normal mouse Normal mouse Normal mouse Heterozygous for gene knock out Breeding to produce a mouse homozygous for the gene knockout

Breeding scheme for producing knockout mice. Blastocysts containing cells, that are both wildtype and knockout cells, are injected into the uterus of a foster mother. This produces offspring that are either wildtype and coloured the same colour as the blastocyst donor (grey) or chimera (mixed) and partially knocked out. The chimera mice are crossed with a normal wildtype mouse (grey). This produces offspring that are either white and heterozygous for the knocked out gene or grey and wildtype. White heterozygous mice can subsequently be crossed to produce mice that are homozygous for the knocked out gene.

There are several variations to the procedure of producing knockout mice-

1. The gene to be knocked out is isolated from a mouse gene library. Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbour sequence, except that it is changed sufficiently to make the gene inoperable. In addition, a second gene, such as herpes tk+, is also included in the construct in order to accomplish a complete selection.
2. Embryonic stem cells are isolated from a mouse blastocyst (a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse.
3. The new sequence from step 1 is introduced into the stem cells from step 2 by electroporation. By the natural process of homologous recombination some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene. The chances of a successful recombination event are relatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes – they are said to be heterozygous. Cells that were transformed with a vector containing the neomycin resistance gene and the herpes tk+ gene are grown in a solution containing neomycin and Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the neomycin resistance gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin.
4. The embryonic stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 1. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant.
5. The knocked-out embryonic stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). These blastocysts are then implanted into the uterus of female mice, where they develop. The newborn mice will therefore be chimeras, some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst.
6. Some of the newborn chimera mice will have gonads derived from knocked-out stem cells, and will therefore produce eggs or sperm containing the knocked-out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked-out gene in all their cells. These mice will be entirely white and are not chimeras, however they are still heterozygous.
7. When these heterozygous offspring are interbred, some of their offspring will inherit the knocked-out gene from both parents; they are homozygous for that allele.  

Lac repressor IPTG complex is a gratuitous inducer of the E.coli Lac operon .Crystals with IPTG were grown in nearly the same condition as though cited above for the native repressor.IPTG concentrations ranging from 1 to 4 times the number of repressor binding sites were used. Though these crystals appears to have nice shape with smoother faces than those grown in the absence of IPTG.

The in vitro Half-Life of the repressor operator complex is 20 min however addition of IPTG reduces the half life 2 4 minut in bacterial culture the beta glactosidase rate of synthesi is maximal 6 min after adding IPTG. Thus IPTG acts directly on the repressor operator complex. physical studies have shown that IPTG binds to the repressor in the absence of DNA but does not change its overall confirmation .

When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars, such as the lac operon. The CAP assists in production in the absence of glucose. CAP is a transcriptional activator that exists as a homodimer in solution, with each subunit comprising a ligand-binding domain at the N-terminus, which is also responsible for the dimerization of the protein and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity and function as allosteric effectors by increasing the protein’s affinity for DNA. CAP has a characteristic helix-turn-helix structure that allows it to bind to successive major grooves on DNA. This opens up the DNA molecule, allowing RNA polymerase to bind and transcribe the genes involved in lactose catabolism. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources. In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes. As cAMP-CAP is required for transcription of the lac operon, this requirement reflects the greater simplicity with which glucose may be metabolized in comparison to lactose.

Fig-Catabolite Activator Protein (CAP) Regulation.

CAP In the absence of CAMP, CAP does not bind the promoter. Transcription occurs at a low rate. Promoter Operator D lacz L lacy lacA RNA Polymerase In the presence of CAMP, CAP binds the promoter and increases RNA polymerase activity. CAMP + CAP Promoter Operator lacz lacY lacA RNA Polymerase

ALLOSTERY-is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site. This is in reference to the fact that the regulatory site of an allosteric protein is physically distinct from its active site.

Allosteric mechanisms of several well-characterized transcriptional regulatory proteins, including the Escherichia coli tryptophan and biotin repressors and the E. coli catabolite repressor protein, involve some degree of ligand-induced folding. In each of these proteins the small molecule acts as a corepressor and its binding promotes DNA binding by promoting folding. Presumably the loss of flexibility accompanying effector binding freezes out conformations that are not productive for binding and/or lowers the entropic penalty for binding.