Who say Slice I mean Splice


Gene splicing is just what it sounds like: cutting the DNA of a gene to add base pairs. Contrary to the immediate image, however, no sharp instruments are involved; rather, everything is done chemically.


Chemicals called restriction enzymes act as the scissors to cut the DNA. Thousands of varieties of restriction enzymes exist, each recognizing only a single nucleotide sequence. Once it finds that sequence in a strand of DNA, it attacks it and splits the base pairs apart, leaving single helix strands at the end of two double helixes. Scientists are then free to add any genetic sequences they wish into the broken chain and, afterwards, the chain is repaired (as a longer chain with the added DNA) with another enzyme called ligase. Hence, any form of genetic material can be spliced together; bacteria and chicken DNA can, and have been, combined. More often, though, splicing is used for important efforts such as the production of insulin and growth hormone to cure human maladies.With modern splicing techniques, enough insulin can be produced for all diabetics. The insulin-producing genes from human DNA are spliced into plasmid DNA; the plasmids are then allowed to infect bacteria, and, as the bacteria multiply, large amounts of harvestable insulin are produced. Splicing has other practical medicinal uses, too. In July of 1996, a 68-year-old woman became the first patient to be treated for arthritis (a disease which affects an estimated 2.1 million Americans) via gene therapy. At the University of Pittsburgh, therapeutic DNA that blocks the production of a specific protein (IL-1) that causes arthritis pain was injected into two of her knuckles.


“The Gene School,” Think Quest; accessed on April 13, 2013.http://library.thinkquest.org/19037/therapy2.html

The Pyruvate Dehydrogenase Complex Is Regulated Allosterically and by Reversible Phosphorylation

Glucose can be formed from pyruvate. However, the formation of acetyl CoA from pyruvate is an irreversible step in animals and thus they are unable to convert acetyl CoA back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to two principal fates: oxidation to CO2 by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid . As expected of an enzyme at a critical branch point in metabolism, the activity of the pyruvate dehydrogenase complex is stringently controlled by several means . High concentrations of reaction products of the complex inhibit the reaction: acetyl CoA inhibits the transacetylase component (E2), whereas NADH inhibits the dihydrolipoyl dehydrogenase (E3). However, the key means of regulation in eukaryotes is covalent modification of the pyruvate dehydrogenase component. Phos-phorylation of the pyruvate dehydrogenase component (E1) by a specific kinase switches off the activity of the complex. Deactivation is reversed by the action of a specific phosphatase. The site of phosphorylation is the transacetylase component (E2), again highlighting the structural and mechanistic importance of this core. Increasing the NADH/NAD+, acetyl CoA/CoA, or ATP/ADP ratio promotes phosphorylation and, hence, deactivation of the complex. In other words, high concentrations of immediate (acetyl CoA and NADH) and ultimate (ATP) products inhibit the activity. Thus,pyruvate dehydrogenase is switched off when the energy charge is high and biosynthetic intermediates are abundant. On the other hand, pyruvate as well as ADP (a signal of low energy charge) activate the dehydrogenase by inhibiting the kinase.


From Glucose to Acetyl CoA. The synthesis of acetyl CoA by the pyruvate dehydrogenase complex is a key irreversible step in the metabolism of glucose.


The complex is inhibited by its immediate products, NADH and acetyl CoA. The pyruvate dehydrogenase component is also regulated by covalent modification. A specific kinase phosphorylates and inactivates pyruvate dehydrogenase, and a phosphatase actives the dehydrogenase by removing the phosphoryl. The kinase and the phosphatase also are highly regulated enzymes.

In contrast, α1-adrenergic agonists and hormones such as vasopressin stimulate pyruvate dehydrogenase by triggering a rise in the cytosolic Ca2+ level, which in turn elevates the mitochondrial Ca2+ level. The rise in mitochondrial Ca2+ activates the pyruvate dehydrogenase complex by stimulating the phosphatase. Insulin also accelerates the conversion of pyruvate into acetyl CoAby stimulating the dephosphorylation of the complex. In turn, glucose is funneled into pyruvate.

The importance of this covalent control is illustrated in people with a phosphatase deficiency. Because pyruvate dehydrogenase is always phosphorylated and thus inactive, glucose is processed to lactic acid. This condition results in unremitting lactic acidosis (high blood levels of lactic acid), which leads to the malfunctioning of many tissues, most notably the central nervous system

Referenced:  “Entry to the Citric Acid Cycle ad Metabolism through it are Controlled,” NCIB, accessed April 04, 2013, http://www.ncbi.nlm.nih.gov/books/NBK22347/.