Specific terminal labeling of DNA molecules.

We report a simple method to directly label or modify a specific terminus of linear DNA molecules. The method is based upon our finding that a presumably triple-stranded structure by RecA-mediated formation at the terminus formed with deoxyoligonucleotides, whose sequence is complementary to the 5' terminus of one of the strands of a double-stranded DNA molecule, is quite stable and can serve as a template for DNA polymerase reaction, with the nucleotides being incorporated by an exchange reaction. This novel type of nucleotide incorporation has made it possible to label a specific terminus of target double-stranded DNA molecules by a direct means (without amplification) regardless of its molecular size, a procedure previously unavailable.

Synaptotagmin XI as a candidate gene for susceptibility to schizophrenia.

Synaptotagmin XI (Syt11) is a member of the synaptotagmin family, which is localized in cells either in synaptic vesicles or the cellular membrane, and is known to act as a calcium sensor. The Syt11 gene is located on chromosome locus 1q21-q22, which was previously reported as a major susceptibility locus of familial schizophrenia. Here, we present evidence for an association between the number of 33-bp repeats in the promoter region of the Syt11 gene and schizophrenia.

Intra-strain polymorphisms are detected but no genomic alteration is found in cloned mice.

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Here, we applied the IGCR procedure to two cloned mice derived from an F1 hybrid of the C57BL/6Cr and DBA/2 strains, in order to investigate the possibility of genomic alteration in the cloned mouse genomes. Each of the five of the genomic alterations we detected between the two cloned mice corresponded to the "intra-strain" polymorphisms in the C57BL/6Cr and DBA/2 mouse strains.

Stable triple-stranded DNA formation and its application to the SNP detection.

We have found that a short stretch (30mer or larger) of triple-stranded DNA structure formed at the terminus (or very near) of linear DNA molecules is unusually stable, withstanding heat treatment at as high as 95 degrees C. The stable triple-stranded structure is formed only when deoxyoligonucleotides are complementary to the strand terminating with 5'-phosphate and not to the strand terminating with 3'-OH. Presence of a single mismatched base in the complementary deoxyoligonucleotides drastically reduces the stability.

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e.

Effects of methylation of non-CpG sequence in the promoter region on the expression of human synaptotagmin XI (syt11).

We have studied the effects of methylation of the promoter region on the expression of human synaptotagmin XI (syt11), a gene implicated in the onset of schizophrenia. Sequence analysis showed that cytosine residues not in the CpG sequence, but still within the promoter region of the gene, are partially methylated. The methylated cytosine residues are located in the mRNA-coding (minus) strand of the promoter region (mCmCTTmCTTmCmC).

Human ATP-binding cassette transporter ABCC10: expression profile and p53-dependent upregulation.

Human ATP-binding cassette (ABC) transporter genes are classified into seven sub-families, where "C" subfamily comprises a total of 13 gene members. The ABCC10 cDNA was cloned in the human full-length cDNA project at the Kazusa DNA Research Institute. However, current information is limited regarding its physiological function and gene expression.

Marking of specific sequences in double-stranded DNA molecules--SNP detection and direct observation.

In this study, we describe a simple method to mark specific sequences in double-stranded DNA molecules. For the marking, we used two specifically designed oligonucleotides, one of which is complementary to the sequence to be marked and the other, serving as a splint, to make the marking stable and detectable by subsequent various analytical means. In the presence of the two deoxyoligonucleotides, whereas RecA protein-mediated reaction converts the sequence to be marked to a regional triple-stranded structure with the complementary (probing) oligonucleotide, DNA ligase transforms it to a stable multi- (possibly quintuple) stranded structure with the splint oligonucleotide.

Isolation of polymorphic mRNAs (cDNAs) by in-gel competitive reassociation.

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Previously IGCR was used to enrich and isolate polymorphic genomic DNA fragments from mouse and human genomic DNA. We have modified the original IGCR procedures specifically for the isolation of polymorphic mRNAs in the form of cDNAs.

Polymorphic 33-bp repeats with promoter-like activity in synaptotagmin 11 gene.

In the immediate upstream region of the transcription initiation site of human synaptotagmin 11 gene, we found a tandem repeat of a 33-bp unit with a promoter-like activity. The tandem repeat is apparently polymorphic in the Japanese population as two DNA samples from 60 schizophrenia patients exhibited an increased number (three) of the unit. In a transfection assay using constructs with different numbers of repeats, the promoter-like activity was found to increase with the number of repeating unit.

Specific cleavage of DNA molecules at RecA-mediated triple-strand structure.

A novel procedure to cleave DNA molecules at any desired base sequence is presented. This procedure is based upon our finding that double-stranded DNA molecules at a site where RecA-mediated triple-stranded DNA structure with a complimentary deoxyoligonucleotide is located can be cleaved by a single-strand specific nuclease, such as nuclease S1 or BAL31, between the first base at the 5' termini of the deoxyoligonucleotides and the nearest base proximal to the 5' termini. Accordingly, the sequence as well as the number of the cleavage sites to be cleaved can be custom designed by selecting deoxyoligonucleotides with specific base sequences for triple-stranded DNA formation.

Screening of gene-associated polymorphisms by use of in-gel competitive reassociation and EST (cDNA) array hybridization.

In-gel competitive reassociation (IGCR) is a method of differential subtraction to enrich polymorphic DNA restriction fragments between two DNA samples without probes or specific sequence information. Here, we show that by combining IGCR and expressed sequence tags (EST) array hybridization, polymorphic DNA fragments associated with genes in complex higher organisms (Arabidopsis thaliana) can be effectively screened, demonstrating that this procedure offers a simple and efficient way to obtain gene-associated polymorphic DNA markers.

Identification of a

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence.