Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

Diagnostic Tests for Primary Renal Hypouricemia

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 μmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.     

Identification of ABCG2 Dysfunction as a Major Factor Contributing to Gout

The ATP-binding cassette, subfamily G, member 2 gene ABCG2/BCRP locates in a gout-susceptibility locus (MIM 138900) on chromosome 4q. Recent genome-wide association studies also showed that the ABCG2 gene relates to serum uric acid levels and gout. Since ABCG2 is also known as a transporter of nucleotide analogs that are structurally similar to urate, and is an exporter that has common polymorphic reduced functionality variants, ABCG2 could be a urate secretion transporter and a gene causing gout. To find candidate mutations in ABCG2, we performed a mutation analysis of the ABCG2 gene in 90 Japanese patients with hyperuricemia and found six non-synonymous mutations. Among the variants, ATP-dependent urate transport was reduced or eliminated in five variants, and two out of the five variants (Q126X and Q141K) were frequently detected in patients. Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. As Q126X and Q141K are a nonfunctional and half-functional haplotype, respectively, their genotype combinations are divided into four estimated functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those who had dysfunctional ABCG2 had an increased risk of gout, and that a remarkable risk was observed in those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). In 2,150 Japanese individuals, the frequency of those with dysfunctional ABCG2 was more than 50%. Our function-based clinicogenetic analysis identified the combinations of dysfunctional variants of ABCG2 as a major contributing factor in Japanese patients with gout.     

ABCG2/BCRP Dysfunction as a Major Cause of Gout

Recent genome-wide association studies showed that serum uric acid (SUA) levels relate to ABCG2/BCRP gene, which locates in a gout-susceptibility locus revealed by a genome-wide linkage study. Together with the ABCG2 characteristics, we hypothesized that ABCG2 transports urate and its dysfunction causes hyperuricemia and gout. Transport assays showed ATP-dependent transport of urate via ABCG2. Kinetic analysis revealed that ABCG2 mediates high-capacity transport of urate (Km: 8.24 ± 1.44 mM) even under high-urate conditions. Mutation analysis of ABCG2 in 90 Japanese hyperuricemia patients detected six nonsynonymous mutations, including five dysfunctional variants. Two relatively frequent dysfunctional variants, Q126X and Q141K, were then examined. Quantitative trait locus analysis of 739 Japanese individuals showed that Q141K increased SUA as the number of minor alleles of Q141K increased (p = 6.60 × 10^?5). Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. Becuase Q126X and Q141K are assigned to nonfunctional and half-functional haplotypes, respectively, their genotype combinations are divided into four functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those with dysfunctional ABCG2 increased the gout risk, especially those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). These genotypes were found in 10.1% of gout patients, but in only 0.9% of control. Our function-based clinicogenetic (FBCG) analysis showed that combinations of the two dysfunctional variants are major causes of gout, thereby providing a new approach for prevention and treatment of the gout high-risk population.     

Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins

Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by “peeling” during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins.     

Targeted exome sequencing of unselected heavy‐ion beam‐irradiated populations reveals less‐biased mutation characteristics in the rice genome

Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M₂ lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M₂ lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations.     

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.