Search

By analyzing 1,780,295 5′-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5 � -end single-pass sequences, cDNAs having the signal sequence were selected by PSORT (‘signal sequence trap’). We also applied ‘secretion or membrane protein-related keyword trap’ based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165 (80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by ‘keyword trap’ preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

In 1999, some Japanese fuel suppliers sold highly concentrated alcohol fuels, which are . mixtures of gasoline and oxygenates, such as alcohol or ether, in amounts of 50% or more. In August 2001, it was reported that some vehicle models using the highly concentrated alcohol fuels encountered fuel leakage and vehicle fires due to corrosion of the aluminum used for the fuel-system parts. The Ministry of Economy, Trade and Industry (METI) and the Ministry of Land, Infrastructure and Transport Government of Japan (MLIT) jointly established the committee on safety for highly concentrated alcohol fuels in September 2001. The committee consisted of automotive technology and metal corrosion experts knowledgeable about preventing such accidents and ensuring user safety. Immersion tests were conducted on metals and other materials used for the fuel-supply system parts to determine the corrosion resistance to each alcohol component contained in the highly concentrated alcohol fuels. It was confirmed that each alcohol component contained in the highly concentrated alcohol fuels currently on the market causes corrosion of the aluminum generally used for automotive fuel-system parts.

The information obtained on sequence variations in the genome (1)(2) could make it possible for clinical science, based on molecular biology, to provide patient-specific medical care based on a patient’s genotype. Of these variations, single-nucleotide polymorphisms (SNPs) are the most common, and they are gradually being revealed as associated with phenotypes related to the onset or severity of diseases and to the effects of drugs. Methods for genotyping SNPs are becoming increasingly important for diagnosing diseases and determining an appropriate treatment for each patient. We recently reported that SNP types can be determined by a new genotyping method based on the bioluminescence detection of the pyrophosphate produced in a specific probe-extension reaction. That method, called bioluminometric assay coupled with modified probe extension reactions (BAMPER) (3), requires that the single-stranded PCR products used as templates for probe-extension reactions be purified before the reaction by use of streptavidin-conjugated magnetic beads to remove the remaining PCR primers and substrates, which is a time-consuming and labor-intensive process. We also reported a BAMPER-based SNP-typing method that uses the PCR products directly and does not require single-stranded DNA (4). Before the SNP-typing probe-extension reaction, exonuclease I and shrimp alkaline phosphatase are used to remove the PCR primers, the residual deoxynucleotide triphosphates, and the residual pyrophosphate produced during the PCR reaction (5) to minimize the background intensity in the subsequent luciferin-luciferase assay. In this report we introduce a simplified and improved BAMPER method that can be used for SNP genotyping of whole blood drawn from individuals as well as for genotyping of purified genomic DNA by use blood dried on paper. The process flow for our method is shown schematically in Fig. 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue8/. The procedure for our novel BAMPER …

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology., An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

Amino acid similarity often needs to be considered in DNA sequence comparison to elucidate gene functions. We propose a Smith-Waterman-like algorithm which considers amino acid similarity and insertions/deletions in sequences at the DNA level and at the protein level in a hybrid manner. The algorithm is applied to cDNA sequences of Oryza sativa and those of Arabidopsis thaliana. The results are compared with the results of application of NCBI's tblastx program (which compares the sequences in the BLAST manner after translation). It is shown that the present algorithm is very helpful in discovering nucleotide insertions/deletions originating from experimental errors as well as amino acid insertions/deletions due to evolutionary reasons.

Although automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10(-17) mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence-collecting yield. The emission and photodestruction cross-sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross-sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence-collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo-destructive, the cross-section of the destruction being about 3.8 x 10(-20) cm2 in buffer solution while that of Texas Red is 1.5 x 10(-21) cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 mW, with an Ar laser (488 nm) for FITC-DNA, and 18 mW, with an He-Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence-collecting efficiency by a factor of 4, and an He-Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra-high sensitivity of 5 x 10(-20) mole/band (S/N-4) was achieved under optimized conditions.

We determined the base sequences of both strands of duplex DNA simultaneously with an automated DNA sequencer. Primers for each strand were labeled with two different dyes and chain extension reactions for both strands produced four DNA families: "A", "C", "G", and "T". An image splitting-prism and band-pass filters were mounted on a real time DNA sequencer to detect fluorescences from the two different dyes separately. As a result, one thousand bases of a clone could be determined at the same time.

We construct a high-precision wavelength meter for infrared tunable diode lasers (TDL) which consists of moving corner reflectors forming a Michelson interferometer, with a single-frequency HeNe laser as a standard. The design of the wavelength meter is described in detail, and possible sources of errors are discussed. An absolute accuracy of 10−7 is demonstrated by measuring CO2 laser wavelengths and almost the same order of accuracy is achieved by applying the wavelength meter to the ν1 band of N2O observed by a TDL.

Vibration-rotation transitions of the fundamental band have been observed for both C35Cl and C37Cl in the 2 Π 1 2 and 2 Π 3 2 states by using an infrared diode laser spectrometer with Zeeman modulation. A few lines of the “hot” band (v = 2 ← 1) have also been recorded for C35Cl. From an analysis of the observed spectra improved values were obtained for the vibrational harmonic frequency and anharmonicity constant, rotational constants, and Λ-doubling parameters. It was found necessary to take into account centrifugal distortion effects on the spin-orbit coupling constant A in the analysis, which gave ( dA dr ) e r e to be −176 ± 38 or −125 ± 38 cm−1, depending upon whether 2Σ− or 2Σ+ states contribute more to the Λ-type doubling. The equilibrium internuclear distance re was calculated from the derived rotational constants to be 1.64506 ± 0.00016 A.

Transient cooling of CO 2 and mixtures of CO 2 –N 2 , CO 2 –He was investigated in the pressure range of range of 5–460 Torr, using different wavelength radiations of the CO 2 laser, i.e. the P and R branches of 10.6 µm and 9.6 µm radiation. Temperature variations of the irradiated gases were detected by a condenser microphone through the pressure changes. Observed experimental results are explained, based on the variation of the vibrational relaxation times of the CO 2 molecule under different physical conditions.

The v = 1 ← 0 vibration-rotation bands of the NS radical in the X 2 Π 1 2 and X 2 Π 3 2 electronic states were observed by using a tunable diode laser. From the least-squares analysis the band origins were determined to be 1204.2755(12) and 1204.0892(19) cm−1, respectively, for X 2 Π 1 2 and X 2 Π 3 2 . The rotational and centrifugal distortion constants and the internuclear distance in the X2Π electronic state were obtained as follows: Be = 0.775549(10) cm−1, De = 0.00000129(33) cm−1, and r e = 1.49403(4) A , with three standard deviations indicated in parentheses.

The ν1 (CO stretching) and ν2 (CF stretching) bands of the FCO radical were observed with Doppler-limited resolution by an infrared diode laser spectrometer with Zeeman and source modulation. The FCO radical was generated by a 60-Hz discharge in one of the following three gas mixtures: O2 + C2F4, CO + SF6, and CO + C2F4, all diluted with He. The observed spectra were analyzed to determine the rotational constants, the centrifugal distortion constants, and the spin-rotation interaction constants. The band origins, 1861.6372(1) and 1026.1283(1) cm−1 [with standard errors in parentheses], which were obtained, were found to agree well with matrix data, 1857 and 1023 cm−1, respectively. The assignment of the observed spectra to the FCO radical was further supported by observing the ν1 band of F13CO, which was obtained from 13CO and SF6. The molecular structure and the force field of FCO are briefly discussed by using molecular constants obtained from the observed spectra.

The vibration-rotation spectrum of the HO2 ν2 bending fundamental band was observed by a semiconductor diode laser spectrometer with a Zeeman modulation technique. The wavelength of the laser was measured by a high-precision λ meter. Of 153 lines which were observed by Zeeman modulation, 137 lines were assigned. A least-squares analysis was carried out on 131 observed lines with 1 ≦ N ≦ 13 and 0 ≦ Ka ≦ 4, to determine the rotational constants, the centrifugal distortion constants, and the spin-rotation interaction constants in the ν2 state. The band origin, which was also derived, is 1391.7540 (2) cm−1 [the value in the parenthesis denotes the standard error]. The force field of the HO2 molecule is briefly discussed using molecular constants obtained in previous works and in the present work.

An infrared photochemical reaction of C 2 F 3 Cl induced by a TEA CO 2 laser was observed. Main reaction products were C 2 F 4 and ClFCCFCl(trans), being quite different from those obtained by thermal or ultraviolet photochemical reactions.

TEA CO2 laser induced chemical reactions of ethylene, chloroethylenes and chloroethanes have been investigated. Since these compounds have absorptions at wavelengths of 10.6 or 9.6 μm band of the CO2 laser, strong excitation of the molecular vibration gives rise to an infrared photochemical reaction. In chlorine derivatives, the cleavage of the C–Cl bond by the laser irradiation appears to initiate decompositions of these materials. The final products were mainly brought about by molecular elimination of HCl, H2, or Cl2. The present results demonstrate that the CO2 laser induced reactions differ form their thermal reactions.

Tunable infrared diode laser spectroscopy has been used to observe a few fine-structure transitions of the He atom in absorption. The helium atom was directly excited by glow discharge in a cell. The 31P1 from 31S0 transition was observed at 1344.5350(4) cm-1, and the three fine-structure components of the 43P2,1,0 from 43S1 transition were found to be resolved and were observed at 918.9270 (3), 918.9368 (3), and 919.0463 (3) cm-1. An additional measurement was also made for the 6F from 5D transition, which appeared at 1344.1404(10) and 1345.2712(10) cm-1 for the singlet and triplet levels, respectively. The values in parentheses denote the experimental error limit and apply to the last digits.

The vibration-rotation spectra of the SF radical were observed by an infrared diode laser spectrometer. The SF radical was generated directly in a cell by glow discharge in a mixture of carbonyl sulfide and carbon tetrafluoride. Zeeman modulation and discharge current modulation were employed to discriminate SF lines from many lines of the reactants and other products. Thirty two lines were assigned to the v = 1 ← 0 and 2 ← 1 bands of both 2 Π 1 2 and 2 Π 3 2 spin states. A least-squares analysis of all the assigned lines provided precise values for molecular parameters in v = 1 and 2 and the two band origins, where ground-state constants were constrained to microwave results. The harmonic frequency ω e was thus determined to be 837.6418(13) cm −1 , and the equilibrium bond length r e was calculated from the B e rotational constant to be 1.596 244(22) A with the error in parentheses, which included 2.5 standard deviations and the uncertainty of Planck's constant.

The poor cold startability is one of the problems in using methanol as the fuel for Otto-type engines. A method for solving this problem is to mix some additives for cold start improvement. The effects of various additives and their concentrations on the improvement of cold startability were studied.

Publisher Summary This chapter focuses on two real-time DNA fragment detection systems, detecting β ray and fluorescence. As visible light produces only a small amount of fluorescence in gels, a visible light-emitting fluorophore is used as a labeling compound. Only one kind of fluorophore is used because of several drawbacks of the multicolor label method. Fluoresceine isothicyanate (FITC) is used as the labeling compound. It gives strong fluorescence and, therefore, a high detecting sensitivity. For a high-sensitivity detection, it is necessary to carry out simultaneous irradiation of all electrophoresis lanes and simultaneous detection of fluorescence from every lane. To achieve this, a side-entry laser irradiation method is employed. It is also used as fluorescence line-image sensing by means of a TV camera system. The real-time fluorescence detection method is a very promising method for rapid DNA sequencing. Its capability is enhanced by improving data acquisition system and electrophoresis condition. A capability of gel reuse is also demonstrated, which is another feature of the fluorescence method.

This paper describes a high-temperature ultrasonic Doppler Velocimetry (HT-UDV) technique that has been successfully applied to measure velocity profiles of the lead-lithium eutectic alloy (PbLi) f...

平成25年度

In a TEA CO2 laser-induced reaction of 1,1-dichloroethane, the relative quantities of the products, C2H2 and ClHC=CH2, were found to vary with the laser wavelength, indicating that the C2H2 molecules are produced by two processes, directly and indirectly from Cl2HC–CH3 within the duration of a single laser pulse.

The mechanism of infrared multiphoton dissociation of C2F3Cl was investigated by observing CO2 laser power dependences of a number of absorbed photon/molecule and reaction rate. A phenomenological model calculation shows that the absorbed energy is localized in two vibrational modes in quasicontinuum state.