Glucose Metabolism Pathway

Amplification parameters were as follows: C, s; C, s; C, s; cycles. Standard curves were made for each experiment, using series of sample dilutions.The exonucleolysis, expected to produce a degradation ladder of the labeled P primer, was analyzed by M urea and PAGE and autoradiography.Reactions were initiated by adding nM pol or pol and incubated for min at C.Oligonucleotides TA, TT were used as template strands.Oligonucleotides D, complementary to the first nucleotides of T, were used as downstream oligonucleotides to construct nucleotide gaps.Oligonucleotide P was labeled at its end with ATP and T polynucleotide kinase.After incubation, reactions were stopped by adding mM EDTA and analyzed by M urea and PAGE and autoradiography.Numbers indicate the amino acid position relative to the N terminus of each DNA polymerase.Invariant residues in the three enzymes aligned are indicated in white letters over a black background.The resulting cDNA is bp in length, with a bp untranslated region, a bp coding sequence, and a bp untranslated region, and it exhibits nucleotide sequence identity with its murine ortholog.The human POLL gene encodes a protein of amino acids with an overall amino acid identity to its murine ortholog.Interestingly, the similarity between human and murine pol is maximal in the pol core, very high in the BRCT domain, and lower in the serineprolinerich domain. Purified human pol was subjected to a glycerol gradient sedimentation. DNA polymerase activity of each fraction was assayed on activated DNA and is expressed as dAMP incorporation. Quantitation of the human pol protein band was carried out by densitometry of the stained gel and is expressed as optical density. However, a more sensitive reverse transcriptionPCR technique detected pol mRNA in every human and murine tissue examined. We therefore performed a quantitative PCR analysis of pol expression in human tissues comparing its expression levels with those of pol, an enzyme thought to have a general role in DNA repair partly because of its housekeeping expression.Relative expression levels of both pol and pol mRNA varied in the different tissues examined, particularly in testis and brain, where pol mRNA levels were clearly higher relative to those of pol. An opposite situation occurs in liver, where the lowest pol: pol ratio was observed.Because pol expression is not only restricted to germinal cells, an additional role of this enzyme in somatic cells must be considered.About of the overproduced pol remained soluble and was precipitated with ammonium sulfate.Interestingly, replacing magnesium ions with manganese when using poly as a substrate stimulated pol and pol polymerization up to fold and fold, respectively.This substratespecific effect could be explained if manganese ions facilitate the slippage capacity of both enzymes.Similar motifs could not be identified in pol sequence, suggesting that, as other family X enzymes, pol has no proofreading activity.To assay whether pol is templatedependent or not, DNA molecules of defined sequence were used as substrates to assay DNA polymerization.Whereas pol was able to perform synthesis on a templateprimer, it was unable to use single stranded DNA or blunt double stranded DNA, thus suggesting that pol is strictly a templatedependent enzyme.

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