Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.     

QTL analysis for resistance to Soybean dwarf virus in Indonesian soybean cultivar Wilis

Soybean dwarf virus (SbDV), a member of the Luteoviridae family, causes serious yield losses in soybean production in northern Japan. We previously found that an Indonesian soybean cv. Wilis had a high level of resistance to SbDV. Although Wilis is infected by SbDV, symptoms are always mild and develop considerably later compared with many susceptible cultivars. To identify the resistance gene(s) to SbDV in Wilis in a quantitative trait loci (QTL) analysis, we used 71 recombinant inbred lines derived from a cross between Wilis and the susceptible Japanese cv. Toyokomachi and a set of published simple sequence repeat (SSR) markers. The SbDV resistance in Wilis was mainly controlled by a single QTL located near the SSR marker Sat_271 on the linkage group A1. This QTL accounted for 79% of the phenotypic variance. A. Uchibori and J. Sasaki equally contributed to this work.     

IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein—a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both α- and β-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.     

C-terminal Elongation of Growth-blocking Peptide Enhances Its Biological Activity and Micelle Binding Affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1–23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1–28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1–28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1–28 GBP undergoes a conformational transition from a random coiled state to an α-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.Growth-blocking peptide (GBP)² was initially identified from the hemolymph of armyworm Pseudaletia separata as a 25-amino acid peptide (1–25 GBP) that prevents the onset of pupation of the host by parasitization of wasp Cotesia kariyai (1–4). Injection of GBP into nonparasitized armyworm larvae early in the last instar delays larval growth and retards pupation for more than a few days. Our previous studies showed that GBP is a hormone-like biogenic peptide of the host armyworm (5, 6). In nonparasitized larvae, the concentrations of GBP were much higher in the early larval stages than in the latter ones. However, parasitization by wasp induces an elevation of GBP in the last larval stages. This elevation was shown to lead to growth retardation via repression of juvenile hormone esterase activity (7–9). Interestingly, a cDNA analysis indicated that the cDNA encodes a 23-amino acid GBP (1–23 GBP), although GBP purified from parasitized armyworm plasma consists of 25 amino acid residues. GBP was expressed as a 23-residue peptide (1–23 GBP) in nonparasitized armyworm larvae, whereas 1–25 GBP, containing Tyr²⁴ and Gln²⁵, was purified from the parasitized larvae. Moreover, the preliminary peptide sequencing of GBP prepared from parasitized larval hemolymph showed the 26^th and 27^th residues on rare occasions (Leu and Ile, respectively) (6). On the basis of these results, we concluded that the TAG stop codon for the 24^th amino acid was unusually decoded as Tyr upon parasitization by parasitoid wasps (10) and predicted that an intact and mature GBP synthesized in the parasitized armyworm larvae would consist of 28 amino acid residues (1–28 GBP).GBP has multiple functions: adhesion and spreading of a specific class of immune cells (plasmatocytes), proliferation of various cultured cells, and induction of larval paralysis (11–13). More than 10 GBP homologous peptides have been identified in Lepidopteran insects, and based on their N-terminal consensus sequences (Glu¹-Asn²-Phe³), they have been categorized as the ENF peptide family (14). The tertiary structure of 1–25 GBP consists of a disordered N-terminal region (residues Glu¹–Gly⁶), a well ordered core region (residues Cys⁷–Thr²²) stabilized by a disulfide bond and a short antiparallel β-sheet, and a short unstructured C-terminal region (Phe²³–Glu²⁵) (15). Because no GBP receptor or its gene has been isolated yet, the nature of either of them at the cellular and molecular levels is poorly understood at present. In contrast, the relationship between the structure and activity of GBP has been well studied by analyzing the biological activities of several variants of GBP and plasmatocyte-spreading peptide (one of the ENF family peptides). Especially, extensive studies on the N termini (residues 1–6) of GBP and plasmatocyte-spreading peptide demonstrated the importance of Phe³ for exerting their hemocyte stimulating activity, thereby suggesting a possible mechanism for receptor activation that requires binding of the aromatic ring of Phe³ and a closely spaced primary amine with receptor activating properties (16–19).In contrast, the C termini of GBP and other ENF peptides have received less attention, because of the weak secondary structure predictions. Therefore, in this study we focused on the C terminus region of GBP and analyzed its contribution to the expression of some biological activities and to the tertiary structure of this peptide. Especially, we prepared GBP with 28 amino acids and characterized the C-terminal region of 1–28 GBP (residues Phe²³–Thr²⁸), because we knew that GBP is present as a 23-amino acid peptide in nonparasitized healthy larvae and that GBP with 28 amino acids has been found only in parasitized host larvae. Our results suggest that the elongation of the C-terminal region of Phe²³–Thr²⁸ greatly reinforced GBP binding with the membrane. Further, the elongation increased GBP inhibition of larval growth.     

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222₁ with unit cell constants of a = 146.8 Å, b = 176.9 Å, and c = 353.7 Å, and the other one had a space group of P2₁2₁2₁ with unit cell constants of a = 209.2 Å, b = 237.5 Å, and c = 299.8 Å. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 Å resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 Å resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.     

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.