Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-Dependent Glycerol Dehydratase

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of Klebsiella pneumoniae, the inactivated enzyme is reactivated in the presence of ATP, Mg2+, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them gdrA and gdrB. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant Escherichia coli cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.     

An Enzyme-Catalyzed Multistep DNA Refolding Mechanism in Hairpin Telomere Formation

Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.     

Development of α1,6-fucosyltransferase inhibitors through the diversity-oriented syntheses of GDP-fucose mimics using the coupling between alkyne and sulfonyl azide

We developed α1,6-fucosyltransferase (FUT8) inhibitors through a diversity-oriented synthesis. The coupling reaction between the fucose unit containing alkyne and the guanine unit containing sulfonyl azide under various conditions afforded a series of Guanosine 5′-diphospho-β-l-fucose (GDP-fucose) analogs. The synthesized compounds displayed FUT8 inhibition activity. A docking study revealed that the binding mode of the inhibitor synthesized with FUT8 was similar to that of GDP-fucose.     

Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.     

Genetic diversity of JC virus in the Saami and the Finns: implications for their population history

The JC virus (JCV) genotyping method was used to gain insights into the population history of the Saami and the Finns, both speaking Finno-Ugric languages and living in close geographic proximity. Urine samples from Saami and Finns, collected in northern and southern Finland, respectively, were used to amplify a 610-bp JCV-DNA region containing abundant type-specific mutations. Based on restriction site polymorphisms in the amplified fragments, we classified JCV isolates into one of the three superclusters of JCV, type A, B, or C. All 15 Saami isolates analyzed and 41 of 43 Finnish isolates analyzed were classified as type A, the European type, and two samples from Finns were classified as type B, the African/Asian type. We then amplified and sequenced a 583-bp JCV-DNA region from the type A isolates of Saami and Finns. According to type-determining nucleotides within the region, we classified type A isolates into EU-a1, -a2, or -b. Most type A isolates from Saami were classified as EU-a1, while type A isolates from Finns were distributed among EU-a1, EU-a2, and EU-b. This trend in the JCV-genotype distribution was statistically significant. On a phylogenetic tree based on complete sequences, most of the type A isolates from Saami were clustered in a single clade within EU-a1, while those from Finns were distributed throughout EU-a1, EU-a2, and EU-b. These findings are discussed in the context of the population history of the Saami and the Finns. This study provides new complete JCV DNA sequences derived from populations of anthropological interest.     

Normotensive glucocorticoid-suppressible hyperaldosteronism in adult

A 40 year-old man was admitted to our hospital for detailed examination of hypokalemia (2.7 mEq/l). His blood pressure was normal. Metabolic alkalosis, ACTH dependent hyperaldosteronism (18 ng/dl) and over-response to synthetic ACTH were observed. Plasma renin activity, on the other hand, was within the normal range (1.7 ng/ml/hr). Serum potassium was normalized to 4.1 mEq/l and the responsiveness of the renin-angiotensin-aldosterone system was recovered after the administration of dexamethasone. These results led us to suggest that this case might be normotensive glucocorticoid-suppressible hyperaldosteronism. The etiology which was not associated with hypertension and low plasma renin activity has not been clarified but may be related to the shortness of duration of this disease. Our case was also afflicted with mild hypercortisolemia and excessive excretion of urinary 17-hydroxycorticosteroid and 17-ketosteroid which was suppressed by the administration of dexamethasone (2 mg/day). These findings may be related to hypersensitivity of the fascicular zone of the adrenal gland to ACTH.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα^−/−, RARα^−/−, and RARγ^−/− cells. RXRα^−/−/ RARα^−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα^−/− cells. In contrast, RXRα^−/−/RARγ^−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.     

Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-D and its aqueous phase behavior

Mannosylerythritol lipids (MELs) produced by yeasts are one of the most promising glycolipid biosurfactants. In this study, we succeeded in the preparation of a novel MEL homolog having no acetyl groups, namely MEL-D. MEL-D was synthesized by lipase-catalyzed hydrolysis of acetyl groups from a known MEL, and identified as 4-O-[2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-(2R,3S)-erythritol. The obtained MEL-D showed a higher critical aggregation concentration (CAC = 1.2 × 10⁻⁵ M) and hydrophilicity compared to known MELs, retaining an excellent surface tension lowering activity (the surface tension at the CAC was 24.5 mN/m). In addition, we estimated the binary phase diagram of the MEL-D–water system based on a combination of visual inspection, polarized optical microscopy, and SAXS measurement. From these results, MEL-D was found to self-assemble into a lamellar (L_α) structure over all ranges of concentration. Meanwhile, the one-phase L_α region of MEL-D was extended wider than those of known MELs. MEL-D might keep more water between the polar layers in accordance with the extension of the interlayer spacing (d). These results suggest that the newly obtained MEL-D would facilitate the application of MELs in various fields as a lamellar-forming glycolipid with higher hydrate ability.