Genome informatics for data-driven biology

A report on the 12th International Conference on Genome Informatics, Tokyo, Japan, 17-19 December 2001.     

Dietary-fiber-degrading enzymes from a human intestinal Clostridium and their application to oligosaccharide production from nonstarchy polysaccharides using immobilized cells

The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum- beijerinckii (isolated from human feces), was investigated. Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source. The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3- beta-glucanase), endo-1,4-beta-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized. These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium. We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system. From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures. In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30-40%.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

High-potential iron-sulfur protein (HiPIP) is the major electron donor to the reaction center complex in photosynthetically growing cells of the purple bacterium Rubrivivax gelatinosus

A gene encoding the high-potential iron-sulfur protein (HiPIP) was cloned from the purple photosynthetic bacterium Rubrivivax gelatinosus. An insertional disruption of this gene by a kanamycin resistance cartridge resulted in a significant decrease in the growth rate under photosynthetic growth conditions. Flash-induced kinetic measurements showed that the rate of reduction of the photooxidized reaction center is greatly diminished in the mutant depleted in the HiPIP. On the other hand, mutants depleted in the low- and high-potential cytochromes c(8), the two other soluble electron carriers, which have been shown to donate an electron to the reaction center in Rvi. gelatinosus, showed growth rates similar to those of the wild type under both photosynthetic and respiratory growth conditions. It was concluded that HiPIP is the major physiological electron donor to the reaction center in Rvi. gelatinosus cells grown under photosynthetic conditions.     

Phylogenetic analysis of the mammalian Hoxc8 non-coding region

The non-coding intergenic regions of Hox genes are remarkably conserved among mammals. To determine the usefulness of this sequence for phylogenetic comparisons, we sequenced an 800-bp fragment of the Hoxc9–Hoxc8 intergenic region from several species belonging to different mammalian clades. Results obtained from the phylogenetic analysis are congruent with currently accepted mammalian phylogeny. Additionally, we found a TC mini satellite repeat polymorphism unique to felines. This polymorphism may serve as a useful marker to differentiate between mammalian species or as a genetic marker in feline matings. This study demonstrates usefulness of a comparative approach employing non-coding regions of Hox gene complexes.     

Melina: motif extraction from promoter regions of potentially co-regulated genes

'Melina' assists users to compare the results of four public softwares for DNA motif extraction in order to both confirm the reliability of each finding and avoid missing important motifs. It is also useful to optimize the sensitivity of software with a series of different parameter settings. AVAILABILITY: Melina is available at http://www.hgc.ims.u-tokyo.ac.jp/Melina/.     

A functional polymorphism in the promoter/enhancer region of the<Emphasis Type="Italic"> FOXP3/Scurfin</Emphasis> gene associated with type 1 diabetes

FOXP3/Scurfin, a member of forkhead/winged-helix proteins, is involved in the regulation of T-cell activation, and essential for normal immune homeostasis. The FOXP3/Scurfin gene is located on chromosome Xp11.23, which includes one of the type 1 diabetes susceptible loci. Therefore, we investigated whether the human FOXP3/Scurfin gene might be a new candidate gene for type 1 diabetes. We first screened the human FOXP3/Scurfin gene for microsatellite and single nucleotide polymorphisms. Next, we performed an association study between the FOXP3/Scurfin gene and type 1 diabetes. Then, the evaluation of promoter/enhancer activity of the intron with (GT)(n) polymorphism was performed by dual luciferase reporter assay. We demonstrated two regions contained microsatellite polymorphisms; one was (GT)(n), located on intron zero and the other (TC)(n) on intron 5, which were under linkage-disequilibrium. The (GT)(15) allele showed a significantly higher frequency in patients with type 1 diabetes than in controls (43.1% vs 32.6%, P=0.0027). The genotype frequencies of (GT)(15)/(GT)(15) in female patients and of (GT)(15) in male patients tended to be higher than those in female ( P=0.064) and male ( P=0.061) controls, respectively. A significant difference in the enhancer activity between (GT)(15) and (GT)(16) dinucleotide repeats was detected. In conclusion, the FOXP3/Scurfin gene appears to confer a significant susceptibility to type 1 diabetes in the Japanese population.     

Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.     

Small open reading frames in 5' untranslated regions of mRnas

Using the 5'-end sequence data from 'oligo-capped' cDNAs, we generated a representative full-length cDNA dataset for 4870 RefSeq entries, and analyzed the 5' untranslated region (UTR) of these genes. To our surprise, about half of the 4870 genes had an upstream ATG before the ATG that starts the longest open reading frame (ORF), suggesting that about half of them have small ORFs in their 5' UTR of average length of 31 amino acids. They require attention for further analysis to identify their biological role.     

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.