Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope.

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

Human T-cell leukemia virus type 2 (HTLV-2) Tax protein transforms a rat fibroblast cell line but less efficiently than HTLV-1 Tax.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are retroviruses with similar biological properties. Whereas HTLV-1 is the causative agent of an aggressive T-cell leukemia, HTLV-2 has been associated with only a few cases of lymphoproliferative disorders. Tax1 and Tax2 are the transcriptional activators of HTLV-1 and HTLV-2, respectively. Here we show that Tax2 transformed a Rat-1 fibroblast cell line to form colonies in soft agar, but the size and number of the colonies were lower than those of Tax1. Use of a chimeric Tax protein showed that the C-terminal amino acids 300 to 353 were responsible for the high transforming activity of Tax1. Activation of cellular genes by Tax1 through transcription factor NF-kappa B is reportedly essential for the transformation of Rat-1 cells. Tax2 also activated the transcription through NF-kappa B in Rat-1 cells, and such activity was equivalent to that induced by Tax1. Thus, the high transforming activity of Tax1 is mediated by mechanisms other than NF-kappa B activation. Our results showed that Tax2 has a lower transforming activity than Tax1 and suggest that the high transforming activity of Tax1 is involved in the leukemogenic property of HTLV-1.

The structure of human metaphase chromosomes: its histological perspective and new horizons by atomic force microscopy.

Studies on the structure of the human chromosome were reviewed from the histological perspective and discussed in connection with our recent findings obtained mainly by atomic force microscopy (AFM). In this paper, we introduce several hitherto known models of the high-order structure of the metaphase chromosome and discuss the actual structure of chromosomes in relation to such structures as spiral chromatids, chromosome bands, and chromosome scaffolds. In chromosomes treated with Ohnuki's hypotonic solution, the chromosome arms were elongated and showed a characteristic spiral pattern of chromatid fibers. On the other hand, alternating transverse ridges and grooves were clearly observed on the surface of chromosomes treated with 0.025% trypsin for G-banding, and these ridges and grooves corresponded to the dark and pale bands of G-banded chromosomes. Similar findings were also found in chromosomes treated with quinacrine mastards for Q-banding. Fibers bridging the gap between the sister chromatids were often observed in G/Q-banded chromosomes; these fibers tended to be restricted within the G/Q-positive portions, suggesting the presence of chromatin fibers bridging these regions. Based on these findings in conjunction with previous studies, we outlined the high-order structure of the human chromosome. Recent advances in nanotechnology have provided new AFM techniques for the imaging and handling of materials at nano-scale resolution. Application of these techniques to chromosome research is expected to provide valuable information on the chromosome structure in relation to its function.