Cloning of the promoters of an Escherichia coli rRNA gene. New experimental system to study the regulation of rRNA transcription.

The promoters of the rrnB gene of Escherichia coli have been cloned on a multicopy, pBR322-derived plasmid by deleting most of the structural part of rrnB and fusing the terminators of the gene immediately to the promoters. Several further deletions were constructed to vary the promoter-terminator distance, destroy or damage selectively any of the promoters or terminators, and vary the distance between the two pairs of P1 P2 and P3 P4 promoters. All these transcription signals were shown to function on the plasmids in vitro and in vivo. The truncated in vivo transcription products initiated at the P1 and P2 promoters of the recombinant plasmids were found to be stable, and the accumulated transcripts could be easily distinguished from the chromosome-coded rRNA. This provides a convenient experimental system to study the regulation of rRNA biosynthesis.

Naturally occurring deuterium is essential for the normal growth rate of cells.

The role of naturally occurring D in living organisms has been examined by using deuterium-depleted water (30-40 ppm D) instead of water containing the natural abundance of D (150 ppm). The deuterium-depleted water significantly decreased the growth rate of the L929 fibroblast cell line, and also inhibited the tumor growth in xenotransplanted mice. Eighty days after transplantation in 10 (59%) out of 17 tumorous mice the tumor, after having grown, regressed and then disappeared. We suggest that the naturally occurring D has a central role in signal transduction involved in cell cycle regulation.

Deuterium depletion can decrease the expression of C-myc Ha-ras and p53 gene in carcinogen-treated mice.

In spite of the fact that the deuterium concentration is over 10 mmol/l in all living organisms, its possible role has been ignored for six decades. Recent studies have shown that the depletion of the naturally occurring deuterium can result in tumour regression in mice, dogs, cats and humans. The effect of deuterium depletion on gene expression plays a key part in tumour development. The carcinogen, 7,12-dimethylbenz(alpha)anthracene (DMBA), was used to increase gene expression in "short term" investigations. The expression of c-myc, Ha-ras and p53 gene was followed in CBA/Ca sensitive inbred mice drinking tap water or deuterium-depleted water (DDW) after induction. By detecting the RNA expression 48 hours after exposure to the carcinogen it was found that the expression of all genes investigated was inhibited in six different organs (spleen, lung, thymus, kidney, liver and lymph node) in the DDW-treated group. It is suggested that genes playing a key role in the cell cycle regulation and tumour development are sensitive to deuterium depletion.

Liposome mediated DNA-transfer into mammalian cells.

We have investigated the interaction of mammalian cells with liposome encapsulated DNA. Tissue cultured mammalian cells were exposed to large, unilamellar phosphatidyl serine liposomes containing DNA molecules from different animal cells or prokaryotic organisms. The liposomes bind rapidly to the surface and are taken up by the cells and significant proportion of the encapsulated DNA is transported to the nuclei. Transient expression of the foreign genetic material could be detected in high percentage of the treated cells for a few days. During this period of time foreign DNA is present in both free and integrated form, however, the free form soon disappears. Stable transformant cell colonies--with continuous expression of new gene(s)--were isolated under selective pressure with a frequency of approx. 10(-5).