Biomedical subjects

R Uchihi

Publications and source records attributed to R Uchihi.

31 records · Page 2Linked to original sources

PubMed1996

Analysis of allelic structures at the D7S21 (MS31A) locus in the Japanese, using minisatellite variant repeat mapping by PCR (MVR-PCR).

To sample the diversity of allelic structures at the D7821 (MS31 A) locus in the Japanese, allele-specific minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) was performed on genomic DNA from a number of Japanese individuals. Three polymorphic positions in the MS31A 5' flanking DNA were typed from 214 un related Japanese, and the distribution of haplotypes was analysed. Allele-specific MVR-PCR using primers that discriminate between different alleles at these polymorphic positions in heterozygous individuals, allows single alleles to be mapped from genomic DNA in approximately 80% of Japanese. 149 Japanese alleles have been mapped to date and all of them, except for two pairs of indistinguishable alleles, have different internal structures. More than half of the mapped alleles showed similar regions of internal structure to other alleles and were classified into groups on this basis.

Alleles↗

PubMed1995

Applications of minisatellite variant repeat (MVR) mapping for maternal identification from remains of an infant and placenta.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to a practical forensic case of an infant and placenta found in an incinerator. They were thought to be left for a few days postmortem, and the infant was severely burnt when found. DNA was extracted from the infantile muscle and maternal placental hematoma. MVR-PCR analysis as well as other common DNA typing (D1S80, HLA-DQA1) were performed on both DNA samples. Both MVR diploid codes were matched although some extra faint bands in the ladder were observed from the maternal placental sample, which probably indicated superimposing of an allele derived only from the mother, and not the infant. In order to detect the original maternal alleles, three flanking polymorphic sites were typed and allele-specific MVR-PCR was performed. Finally, one maternal allele not inherited by the infant and two alleles from the infant were typed. Two alleles suggested the infant and/or mother was Japanese. The two diploid codes including one possibly from the mother were deduced and compared with other codes in the databases for evaluating the discriminating power.

Base Sequence↗

PubMed1994

DNA typing of the D1S8 (MS32) locus by rapid detection minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) assay.

The typing of the D1S8 (MS32) locus using the minisatellite variant repeat (MVR) polymerase chain reaction (PCR) method was performed by visualising amplified DNA stained with ethidium bromide. The results from rapid detection MVR-PCR were compared with those from the original MVR-PCR using Southern blot hybridisation with a 32P-labelled probe. With genomic DNA extracted from blood samples of 40 healthy unrelated Japanese individuals, the first 41 codes, on average, were correctly determined by rapid detection MVR-PCR without band intensity information, compared with at least 60 codes typed by the original MVR-PCR. The rapid detection MVR-PCR method was applied to bloodstains to simulate forensic samples. On average, 39 code positions could be determined from DNA extracted from 3-month-old bloodstains of six persons. Rapid detection MVR-PCR is more convenient than the original MVR-PCR, furnishes much information with regard to personal identification, and should be applicable to forensic fields.

Alleles↗

PubMed1994

Efficient amplification of the HLA-DQA1 gene in single genomes using a semi-nested polymerase chain reaction.

A semi-nested polymerase chain reaction (PCR) was introduced to amplification of the HLA-DQA1 gene, which is a single-copy gene, in a single genome. The limitation of template DNA for genotyping is 1 ng of genomic DNA, or more in the case of ordinary PCR. When reamplification with the same primers was performed, primer dimer was generated and the sensitivity was not improved. We designed a semi-nested primer for the second round of PCR, using the semi-nested PCR, more than 3 pg of template DNA could be amplified and typed. Furthermore, this method was applied to amplify DQA1 gene in single human sperm having haploid DNA, and followed by typing with sequence-specific oligonucleotide (SSO) probes. The semi-nested PCR technique was found to enhance the sensitivity of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.

Base Sequence↗

PubMed1994

Potential forensic applications of minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to samples from various human tissues. All DNA samples obtained from an individual's organs at autopsy consistently gave the same digital diploid codes. Even 1 ng of genomic DNA was sufficient to obtain authentic diploid MVR coding ladders. MVR-PCR could be reliably applied to DNA isolated from bloodstains, saliva stains, seminal stains and plucked hair roots, and should become a powerful tool for individual identification in forensic investigations.

Base Sequence↗

PubMed1993

[DNA typing of the three HLA-class II loci from saliva stains].

The DNA typing of the HLA-class II loci, HLA-DPB1, HLA-DQA1 and HLA-DRB, from saliva stains is described. In addition to the experimentally prepared saliva stains, a stain on a towel in a burglar case was subjected to the analysis. Using 10 ng of the extracted DNA, we could type all the three loci with all of the specimens tested. The incidence of the saliva stain in the practical case was estimated to be 3.18 x 10(-5) in Japanese population. We found that the aging of saliva stains at least six months did not significantly influence the DNA typing.

DNA↗

PubMed1993

Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction.

The DNA typing of HLA-DQA1 from bloodstains is described. Although amplification of DNA from bloodstains was sometimes difficult, the addition of BSA to the PCR medium induced good amplification. When a very limited amount (2 mm2, equivalent to 0.5 microliter blood) of bloodstain was analysed, the amplification was difficult. However, the addition of salmon sperm DNA to the DNA isolation medium, in the amount of 1 microgram per tube, yielded sufficient PCR products even when such a small amount of extract was analysed. DNA from bloodstains aged up to one year could be sufficiently amplified, if salmon sperm DNA was added to the DNA isolation medium, and BSA to the PCR medium.

Animals↗

PubMed1992

[DNA typing and analysis of the D1S8 (MS 32) allele in the Japanese population by the minisatellite variant repeat (MVR) mapping by polymerase chain reaction (PCR) assay].

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) of the D1S8 (MS32) locus from total genomic DNA and size separated alleles in the Japanese population was performed. DNA was extracted from blood in healthy non-related Japanese individuals. The digital data obtained were computer analysed, and from this single assay all unrelated individuals tested could be unambiguously distinguished. The average difference in diploid codes between unrelated individuals was 36 exclusions over the first 60 repeat unit positions. The mean proportions of a-, t-, and 0-type repeat units were 73.5%, 24.5% and 2.1%, respectively. MVR-PCR gives digital profiles which show extreme levels of individual variation and should prove very useful for personal identification. The advantages and further applications of MVR-PCR are also discussed.

Alleles↗

PubMed1992

Deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese.

The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.

Asian People↗

PubMed1991

Frequency of HLA-DQA1 alleles in the Japanese population.

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.

Alleles↗

PubMed1989

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.

Amino Acid Sequence↗

PubMed1988

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.

Amino Acid Sequence↗

PubMed1986

Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47: complete nucleotide sequence of the outer wall protein gene.

Bacillus brevis 47 contains two cell wall proteins termed the outer wall protein (OWP) and middle wall protein (MWP), each of which forms hexagonal arrays in the cell wall. A 6-kilobase BglII-BclI fragment of B. brevis 47 DNA cloned into Bacillus subtilis with a derivative of pHW1 as a vector directed the synthesis of a polypeptide, with almost the same molecular weight as the authentic OWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was specifically recognized by the anti-OWP antibody. Nucleotide sequence analysis of the subfragment revealed that it contains two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the MWP, and the downstream open reading frame corresponds to the entire translational portion of the OWP. The latter encodes a secretory precursor of the OWP, consisting of a total of 1,004 amino acid residues with a signal peptide of 24 amino acid residues at its amino-terminal end. Futhermore, analysis of transcripts in B. brevis 47 suggests that the MWP and OWP genes, in that order, constitute a cotranscriptional unit and that the major promoter shared by the two genes is located upstream of the MWP gene.

Amino Acid Sequence↗