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. The whole marking process is simple and completed in a single reaction mixture. Because RecA protein makes the marking to proceed with high fidelity, we were able to mark (detect) SNPs in complex genomes like human's. Furthermore, the structure of the marked sequence is stable and quite distinct enough to be readily detectable by biochemical means or direct observation by scanning probe microscopy.

Isolation and characterization of mouse homolog of the neutrophil activating peptide-2.

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.