Nuceotide metabolism: Answer Set

Since E. coli have a single carbamoyl phosphate synthase that is used for both de novo pyrimidine biosynthesis and arginine biosynthesis, it would not be practical for E.coli to regulate pyrimidine biosynthesis through this enzyme (otherwise large amounts of UMP would not only shut off de novo pyrimidine biosynthesis but also arginine biosynthesis). Instead E.coli choose to regulate the second step in pyrimidine biosynthesis, aspartate transcarbamylase. Increasing or decreasing the levels of this enzyme activity would influence flux through the de novo pyrimidine pathway but not any other pathways.

Since animal cells have two different carbamoyl phosphate synthases, they can regulate the de novo pyrimidine pathway by making the cytosolic enzyme responsive to final products of pyrimidince metabolism. Since the mitochondrial carbamylphosphate synthase (dedicated to arginine synthesis) is unresponsive to pyrimidine nucleotides, it can continue to make carbamoyl phosphate for arginine and urea synthesis.

Rationale behind each step in a yeast gene replacement strategy:

YEAST LACKING OROTIDYLATE DECARBOXYLASE ARE MAINTAINED ON URACIL MEDIUM:

Lack of orotidylate decarboxylase prevents these yeast from making UMP through the de novo pyrimidine pathway. These cells would die in the absence of some form of preformed pyrimidine. These yeast will grow in uracil medium. Uracil can be phosphoribosylated to UMP. No decarboxylation is necessary to make UMP.

TRANSFORMATION OF YEAST WITH A PLASMID CONTAINING "A GENE OF INTEREST" AND A URA3 GENE:

Such transformation (and integration) events can be selected for by growing these yeast without uracil. Parent cells that fail to take-up plasmid lack orotidylate decarboxylase so they die in the absence of uracil. Cells that take-up the plasmid now gain a copy of the Ura3 gene and orotidylate decarboxylase activity and thus can make UMP through the de novo pyrimidine pathway.

Site-specific recombination at "the gene of interest" results in two copies of the gene of interest separated by the plasmid sequence (incuding the Ura3 gene).

A SECOND RECOMBINATION CAUSES LOSS OF PLASMID:

This also causes the cells to again loose orotidylate decarboxylase function. One can perform a "negative selection" selection for such loss of function events. Cells are supplied with a toxic analog of orotate, 5-fluoroorotate. This would be converted to 5-fluoroorotidylate (5-FOMP) in all cells but whether subsequent metabolism occurs or not depends on whether the plasmid is still present in the cell or whether it is lost. Cells lacking Ura3 would not be able to decarboxylate 5-FOMP and thus toxic fluorinated compounds such as 5-FdUMP would not be made in Ura3- cells. In contrast, yeast cells that did not go through a second recombination would continue to have orotidylate decarboxylase activity and convert 5-fluoroorotate to toxic nucleotides and the cell would die.

TRIMETHOPRIME:

This compound is a small molecular weight inhibitor of bacterial dihydrofolate reductase. Inhibition of dihydrofolate reductase (DHFR) depletes cells of tetrahydrofolate and this in turn causes inhibition of thymidylate synthase, accumulation of dUTP, DNA strand fragmentation and cell death.

The toxicity of trimethoprime is selective because the compound binds bacterial DHFR about 1,000 times more tightly than the mammalian cells.

ACYCLOVIR (Acylcogluanosine):

This compound causes DNA and RNA chain termination in cells infected with herpes virus. The compound has the equivalence of a 5'- OH and can be incorporated into a growing nucleic acid polymer. However, acyclovir lacks the equivalent of the 3'-OH, so after it is incorporated into a growing polymer, no other nucleotides can be added to the acyclovir nucleotide.

The toxicity of acyclovir is selective against Herpes infected cells because initial activation of acyclovir to a phosphorylated nucleotide can only be carried out by Herpes thymadine kinase (with broad substrate specificity). Mammalian salvage enzymes do not act acyclovir so the compound exists in a non-toxic form in non-infected mammalian cells.