Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli

The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence. Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson–Crick base pairing. However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the λ phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product. Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e. TGGE analysis of random PCR products). The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb). The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences. The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above-mentioned ideas as rational. We believe that this is the first case to computer-utilize a genome sequence as a whole.     

Species-identification dots: a potent tool for developing genome microbiology

Identification of species has long been done by phenotype-based methodologies. Recently, genotype-based species identification has been shown to be possible by way of Genome profiling, which is based on a temperature gradient gel electrophoresis (TGGE) analysis of random PCR products. However, the results, though sufficient in information, provided by genome profiling were complicated and difficult to deal with objectively. To cope with this, a technology of utilizing species identification dots (spiddos), which corresponds to structural transition points of DNAs, was introduced. Pattern similarity score (PaSS), derived from spiddos, was shown to be usable for quantitatively measuring the closeness between genomes. This was demonstrated with the experiments applied to the genomes of Escherichia coli O157:H7 (19 strains). The same genomes were also examined by sequencing and RFLP methods in order to compare the effectiveness of these three methods. As a result, the spiddos method was shown to give reasonable results and to be the most advantageous for measuring the closeness between species in general. This means that spiddos is pushing the heavy gate open for genome microbiology.     

Characterization of three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II isolated from the green alga, Dunaliella salina

Three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II (LHC II) were isolated from the thylakoid membranes of Dunaliella salina grown under different irradiance conditions. Cells grown under a low intensity light condition (80 micromol quanta m(-2) s(-1)) contained one form of LHC II, LHC-L. Two other forms of LHC II, LHC-H1 and LHC-H2, were separated from the cells grown under a high intensity light condition (1,500 micromol quanta m(-2) s(-1)). LHC-L and LHC-H1 showed an apparent particle size of 310 kDa and contained four polypeptides of 31, 30, 29 and 28 kDa. LHC-H2, with a particle size of 110 kDa, consisted of 30 and 28 kDa polypeptides. LHC-L contained 7.5 molecules of Chl a, 3.2 of Chl b and 2.1 of lutein per polypeptide, analogous to the content in higher plants. LHC-H1, with 5.6 molecules of Chl a, 2.5 of Chl b and 1.8 of lutein per polypeptide was similar to that in the green alga Bryopsis maxima. LHC-L and LHC-H1 maintained high efficiency energy transfer from Chl b and lutein to Chl a molecules. LHC-H2 showed a high Chl a/b ratio of 7.5 and contained 3.4 molecules of Chl a, 0.5 of Chl b and 1.4 of lutein per polypeptide. Chl b and lutein could not completely transfer the excitation energy to Chl a in LHC-H2.     

Liver autophagy contributes to the maintenance of blood glucose and amino acid levels

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.     

A Novel dnaJ Family Gene,sflA, Encodes an Inhibitor of Flagellation in Marine Vibrio Species

The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ΔflhF mutant makes no flagellum, whereas the ΔflhFG double-deletion mutant usually lacks a flagellum. However, the ΔflhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ΔflhFG strain (ΔflhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of ΔflhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the ΔflhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall.     

Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous Retroviruses

The host defense against viral infection is acquired during the coevolution or symbiosis of the host and pathogen. Several cellular factors that restrict retroviral infection have been identified in the hosts. Feline leukemia virus (FeLV) is a gammaretrovirus that is classified into several receptor interference groups, including a novel FeLV-subgroup D (FeLV-D) that we recently identified. FeLV-D is generated by transduction of the env gene of feline endogenous gammaretrovirus of the domestic cat (ERV-DCs) into FeLV. Some ERV-DCs are replication competent viruses which are present and hereditary in cats. We report here the determination of new viral receptor interference groups and the discovery of a soluble antiretroviral factor, termed Refrex-1. Detailed analysis of FeLV-D strains and ERV-DCs showed two receptor interference groups that are distinct from other FeLV subgroups, and Refrex-1 specifically inhibited one of them. Refrex-1 is characterized as a truncated envelope protein of ERV-DC and includes the N-terminal region of surface unit, which is a putative receptor-binding domain, but lacks the transmembrane region. Refrex-1 is efficiently secreted from the cells and appears to cause receptor interference extracellularly. Two variants of Refrex-1 encoded by provirus loci, ERV-DC7 and DC16, are expressed in a broad range of feline tissues. The host retains Refrex-1 as an antiretroviral factor, which may potentially prevent reemergence of the ERVs and the emergence of novel ERV-related viruses in cats. Refrex-1 may have been acquired during endogenization of ERV-DCs and may play an important role in retroviral restriction and antiviral defense in cats.     

Optimization of conditions for thermal treatment of rice bran using an accelerator including an organo-iron compound

A method for thermal conversion of raw organic waste (ROW) to a compost-like material (CLM) with higher levels of unsaturated carbohydrates, nitrogen- and oxygen-containing compounds was developed, in which rice bran and an organo-iron compound were employed as a model ROW and the accelerator, respectively. To evaluate the qualities of CLMs, organic substances of an acid insoluble fraction of alkaline extracts (AIAEs) from a CLM were structurally characterized by elemental analysis, pyrolysis-gas chromatography/mass spectrometry and FT-IR. The levels of unsaturated carbohydrates, and nitrogen- and oxygen-containing compounds in the CLM samples were increased by long-term treatment (60°C for 5 days, 170°C for 3 days). In particular, the high lipid content of the AIAEs, which was indicative of inadequate digestion of CLM components, was dramatically reduced in the presence of the accelerator.     

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.