Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor)

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K m for both substrates, a normal V max , a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T→G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5′-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47622/1/439_2004_Article_BF00291707.pd

Deep Sequencing the Transcriptome Reveals Seasonal Adaptive Mechanisms in a Hibernating Mammal

Mammalian hibernation is a complex phenotype involving metabolic rate reduction, bradycardia, profound hypothermia, and a reliance on stored fat that allows the animal to survive for months without food in a state of suspended animation. To determine the genes responsible for this phenotype in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) we used the Roche 454 platform to sequence mRNA isolated at six points throughout the year from three key tissues: heart, skeletal muscle, and white adipose tissue (WAT). Deep sequencing generated approximately 3.7 million cDNA reads from 18 samples (6 time points ×3 tissues) with a mean read length of 335 bases. Of these, 3,125,337 reads were assembled into 140,703 contigs. Approximately 90% of all sequences were matched to proteins in the human UniProt database. The total number of distinct human proteins matched by ground squirrel transcripts was 13,637 for heart, 12,496 for skeletal muscle, and 14,351 for WAT. Extensive mitochondrial RNA sequences enabled a novel approach of using the transcriptome to construct the complete mitochondrial genome for I. tridecemlineatus. Seasonal and activity-specific changes in mRNA levels that met our stringent false discovery rate cutoff (1.0×10−11) were used to identify patterns of gene expression involving various aspects of the hibernation phenotype. Among these patterns are differentially expressed genes encoding heart proteins AT1A1, NAC1 and RYR2 controlling ion transport required for contraction and relaxation at low body temperatures. Abundant RNAs in skeletal muscle coding ubiquitin pathway proteins ASB2, UBC and DDB1 peak in October, suggesting an increase in muscle proteolysis. Finally, genes in WAT that encode proteins involved in lipogenesis (ACOD, FABP4) are highly expressed in August, but gradually decline in expression during the seasonal transition to lipolysis

CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands1, 2, 3, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before4, 5, 6, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC)7, 8, 9, 10. Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues11