Sequence requirements for the termination of rolling-circle replication of plasmid pT181

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.     

Retrofitting YACs for direct DNA transfer into plant cells

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.     

Distinct response of Medicago suspension cultures and roots to Nod factors and chitin oligomers in the elicitation of defense-related responses

The induction of plant defense-related responses by chitin oligomers and the Rhizobium meliloti lipo-chito-oligosaccharide nodulation signals (Nod factors) in Medicago cell cultures and roots was investigated by following the expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway, such as chalcone synthase, chalcone reductase, isoflavone reductase, as well as genes encoding a pathogenesis-related protein and a peroxidase. In suspension-cultured cells, all genes except the peroxidase gene were induced by both the R. meliloti Nod factor NodRm-IV(C16:2,S) and chitin oligomers with a minimum of three sugar residues. However, activation of these genes was not elicited by the symbiotically inactive, desulfated NodRm-IV(C16:2). Moreover, the cells were more sensitive to the chitin oligosaccharides than to the Nod factor. Analysis of flavonoids in Medicago microcallus cultures revealed differences between cells treated with N -acetyl-chitotetraose and those treated with Nod factor and demonstrated increased production of the phytoalexin medicarpin in the presence of Nod factor. In Medicago roots, none of the tested genes was activated by the N -acetylchitotetraose, whereas the Nod factor at micro-molar concentration enhanced transient expression of the isoflavonoid biosynthetic genes. The differential responses to Nod factors and chitin oligomers suggest that Medicago cells possess distinct perception systems for these related molecules.     

Lectin histochemical HPA-binding pattern of human breast and colon cancers is associated with metastases formation in severe combined immunodeficient mice

Metastasis formation is a major clinical problem in cancer treatment, and no significant progress in the treatment of metastatic spread has been made. This apparent lack of progress is partly caused by the absence of clinically relevant animal models of meta stases. The binding of the lectin Helix pomatia agglutinin (HPA) has been associated with a poor prognosis in breast and colon cancer patients. HPA-positive and -negative human breast and colon cancer cell lines were transplanted into severe combined immunodeficient (SCID) mice. HPA-positive breast cancer cell lines (MCF-7 and T47D) metastasized in SCID mice, whereas the HPA-negative ones (BT20, HS578T and HBL100) did not. The HPA-positive colon cancer cell line HT29 metastasized, while the HPA-negative ones (COLO320DM, SW480 and SW620) did not. However, in two of eight SCID mice inoculated with the HPA-negative colon cancer cell line, CACO2 metastatic deposits were found. Despite this exception, HPA binding is a good indicator of the metastasis of human breast and colon cancer cells in SCID mice: 23 out of 26 HPA-positive cancers metastasized, as opposed to only two out of 38 HPA-negative cancers. This experimental model is well suited for investigating the functional role of carbohydrate residues recognized by HPA in breast and colon cancer metastasis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.