Identification of a Novel β-Cell Glucokinase (GCK) Promoter Mutation (−71G>C) That Modulates GCK Gene Expression Through Loss of Allele-Specific Sp1 Binding Causing Mild Fasting Hyperglycemia in Humans

OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: The beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: A novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (> or = 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS: A novel beta-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included

Characterization and crystal structure of the type IIG restriction endonuclease RM.BpuSI

A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x15-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R–M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis

Highlights of the DNA cutters:a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine

Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes

Voyage helvetique a travers les caracteristiques cliniques et genetiques du diabete du sujet jeune. [Swiss journey through the clinical and genetic characteristics of diabetes in young patients]

The aim of this study is to understand better the genetic causes of type II diabetes and the phenotypic consequences of the genetic changes. We first investigated the relative prevalence of the different forms of diabetes in young adults and their clinical features. 51 non-obese patients were identified in whom diabetes had been diagnosed before age 40; cases of typical insulin-dependent type I diabetes were excluded. A search for mutations of the glucokinase and HNF-1 alpha genes and for mitochondrial DNA was made, anti-islet and anti-GAD antibodies were determined and HLA class II genotyping was performed. Patients were subdivided on clinical grounds into a MODY (maturity onset diabetes of the young) group (n = 19) and a non-MODY group (n = 32). MODY is a form of diabetes which has an autosomal dominant inheritance for which 3 genes have already been implicated (MODY1, HNF-4 gene; MODY2, glucokinase gene, and MODY3, HNF-1 alpha gene). In the MODY group we identified 3 patients with MODY2, 1 with MODY3, 1 with the 3243 mitochondrial mutation and a further patient with autoimmune diabetes. In the non-MODY group we found 5 patients with autoimmune diabetes and 1 with MODY2. No clinical parameter was helpful in classifying patients in one of these subclasses of diabetes; however, glucagon stimulated C-peptide was useful in discriminating between MODY2 patients and the others. Young and lean non-insulin-dependent diabetic patients thus constitute a very heterogeneous group, though presenting similar clinical features. In the second study we analyzed hepatic glucose metabolism in patients with a mutation of the glucokinase gene expressed in both liver and islet beta cells. We found that endogenous glucose production is inadequately inhibited by hyperglycemia, a fact which contributes to the pathogenesis of hyperglycemia in these patients

PCR amplification of Ureaplasma urealyticum in respiratory secretions of an HIV positive patient

PCR with Mycoplasma primer were applied to detect Mycoplasma Spp. and Ureaplasma urealyticum in respiratory secretions of HIV infected and AIDS patients