No abstract

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli #-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from Pgalactosidase, detected by radioimmunoassay and purified. Complete purification of the A chain and partiaf purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.Recently improved methods of DNA chemical synthesis, combined with recombinant DNA technology, permit the design and relatively rapid synthesis of modest-sized genes that can be incorporated into prokaryotic cells for gene expression. The feasibility of this general approach was first demonstrated by the synthesis, and expression in Escherichia col, of a gene for the mammalian peptide somatostatin (1).Following the precursor protein approach used for somatostatin (1), the experimental design for this work was such that the insulin peptide chains would be made in vio as short tails joined by a methionine to the end of ,3-galactosidase. After synthesis, the insulin chains, which contain no methionine, can be cleaved off efficiently by treatment with cyanogen bromide. We deliberately chose to construct two separate bacterial strains, one for each of the two peptide chains of insulin: the 21-amino-acid A chain and the 30-amino-acid B chain. In native insulin, the two chains are held together by two disulfide bonds, and methods have been available for years for joining the chains correctly, in vitro, by air oxidation (2). The efficiency of correct joining has been variable and often low. However, by using S-sulfonated derivatives and an excess of A chain, 50-80% correct joining has been obtained (3).The synthetic plan and chemical synthesis of the DNA fragments coding for the A and B chains of human insulin were described in a previous paper (4) and were summarized in Fig. 1 Enzymes and DNA Preparations. T4 DNA ligase and T4 polynucleotide kinase were purified as described (6). Restriction endonuclease EcoRI was purified by the procedure of Greene et al. (7); HindIII was purified by a method developed by D. Goeddel (unpublished). Restriction endonuclease BamHI was purchased from Bethesda Research (Rockville, MD); E. coli alkaline phosphatase was purchased from Worthington.Plasmids, including pBR322 (8), were isolated by a published procedure (9) with some modifications. The chemical synthesis of the deoxyoligonucleotides (figure 1 of ref. 4) has been described (4). Xplac5 DNA was isolated as described (10).The following reaction buffers were used: kinase buffer, 60 mM Tris-HCl, pH 8/15mM 2-mercaptoethanol/10 mM MgCl2; ligase buffer, 20mM Tris-HCl, pH 7.5/10mM dithiothreitol/10 mM MgCl2; BamHI buffer, 20 mM Tris-HCl, pH 7.5/7 mM MgCl2/2 mM 2-mercaptoethanol; EcoRI-HindIll buffer, BamHI buffer containing 50 mM NaCl; and phosphatase buffer, 50 mM Tris-HCl, pH...

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term followup Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.T he landmark Diabetes Control and Complications Trial (DCCT; 1983-1993 clearly showed that intensive (INT) glycemic control profoundly reduces the development and progression of microvascular complications in type 1 diabetes (T1D). The DCCT participants were subsequently followed in the Epidemiology of Diabetes Interventions and Complications (EDIC) Study (1994 to present), during which all subjects were advised to practice INT treatment. Surprisingly, those previously assigned to conventional (CONV) therapy continued to develop complications, such as nephropathy, retinopathy, and macrovascular diseases, at significantly higher rates than the previous INT therapy group, despite nearly similar HbA1c levels during the EDIC Study (1-3). This persistence of benefit from early application of INT therapy, called "metabolic memory," is an enigma in the field of T1D: recent studies have suggested the involvement of epigenetic mechanisms (4-9).Epigenetics is the study of mostly heritable changes in gene expression and phenotype that occur without alterations in the underlying DNA sequence. Epigenetic states are affected by environmental factors, such as aberrant nutrition and metabolic states (4, 6-8, 10). DNA methylation (DNA-me; the classic epigenetic mark) and posttranslational modifications (PTMs) of histo...

No abstract

No abstract