Novel mutation at the initiation codon in the Norrie disease gene in two Japanese families

We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.     

TiLIA: a software package for image analysis of firefly flash patterns

As flash signaling patterns of fireflies are species specific, signal‐pattern analysis is important for understanding this system of communication. Here, we present time‐lapse image analysis (TiLIA), a free open‐source software package for signal and flight pattern analyses of fireflies that uses video‐recorded image data. TiLIA enables flight path tracing of individual fireflies and provides frame‐by‐frame coordinates and light intensity data. As an example of TiLIA capabilities, we demonstrate flash pattern analysis of the fireflies Luciola cruciata and L. lateralis during courtship behavior.     

Codon bias differentiates between the duplicated amylase loci following gene duplication in Drosophila

We examined the pattern of synonymous substitutions in the duplicated Amylase (Amy) genes (called the Amy1- and Amy3-type genes, respectively) in the Drosophila montium species subgroup. The GC content at the third synonymous codon sites of the Amy1-type genes was higher than that of the Amy3-type genes, while the GC content in the 5'-flanking region was the same in both genes. This suggests that the difference in the GC content at third synonymous sites between the duplicated genes is not due to the temporal or regional changes in mutation bias. We inferred the direction of synonymous substitutions along branches of a phylogeny. In most lineages, there were more synonymous substitutions from G/C (G or C) to A/T (A or T) than from A/T to G/C. However, in one lineage leading to the Amy1-type genes, which is immediately after gene duplication but before speciation of the montium species, synonymous substitutions from A/T to G/C were predominant. According to a simple model of synonymous DNA evolution in which major codons are selectively advantageous within each codon family, we estimated the selection intensity for specific lineages in a phylogeny on the basis of inferred patterns of synonymous substitutions. Our result suggested that the difference in GC content at synonymous sites between the two Amy-type genes was due to the change of selection intensity immediately after gene duplication but before speciation of the montium species.     

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.     

Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src

Reactive oxygen species are involved in the mitogenic signal transduction cascades initiated by several growth factors and play a critical role in mediating cardiovascular diseases. Interestingly, H(2)O(2) induces tyrosine phosphorylation and trans-activation of the platelet-derived growth factor receptor and the epidermal growth factor receptor in many cell lines including vascular smooth muscle cells. To investigate the molecular mechanism by which reactive oxygen species contribute to vascular diseases, we have examined a signal transduction cascade involved in H(2)O(2)-induced platelet-derived growth factor receptor activation in vascular smooth muscle cells. We found that H(2)O(2) induced a ligand-independent phosphorylation of the platelet-derived growth factor-beta receptor at Tyr(1021), a phospholipase C-gamma binding site, involving the requirement of protein kinase C-delta and c-Src that is distinct from a ligand-dependent autophosphorylation. Also, H(2)O(2) induced the association of protein kinase C-delta with the platelet-derived growth factor-beta receptor and c-Src in vascular smooth muscle cells. These findings will provide new mechanistic insights by which enhanced reactive oxygen species production in vascular smooth muscle cells induces unique alleys of signal transduction distinct from those induced by endogenous ligands leading to an abnormal vascular remodeling process.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.     

A new DNA combing method for biochemical analysis

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, λDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.