Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.     

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import,processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.     

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.     

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.