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.     

Amplification of the amoA gene from diverse species of ammonium-oxidizing bacteria and from an indigenous bacterial population from seawater.

Because the chemolithotrophic ammonium-oxidizing bacteria are an integral component of nitrogen biogeochemistry, a sensitive and accurate method to detect this ecologically important group of microorganisms is needed. The amoA gene of these organisms encodes the active site of ammonia monooxygenase, an enzyme unique to this group of nitrifying bacteria. We report here the use of the PCR technique to detect the amoA gene from pure cultures of chemolithotrophic ammonium-oxidizing bacteria, ammonium oxidizers introduced into filtered seawater, and the natural bacterial population of an unfiltered seawater sample. Oligonucleotide primers, based on the published amoA sequence from Nitrosomonas europaea, were used to amplify DNA from pure cultures of Nitrosomonas europaea, Nitrosomonas cryotolerans, and Nitrosococcus oceanus and from bacteria in seawater collected offshore near the Florida Keys. Partial sequencing of the amplification products verified that they were amoA. These primers, used in conjunction with a radiolabeled amoA gene probe from Nitrosomonas europaea, could detect Nitrosococcus oceanus inoculated into filter-sterilized seawater at 10(4) cells liter-1. Native marine bacteria containing amoA could also be detected at their naturally occurring titer in oligotrophic seawater. Amplification of the gene for ammonia monooxygenase may provide a method to estimate the distribution and relative abundance of chemolithotrophic ammonium-oxidizing bacteria in the environment.     

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.     

Atomic and residue hydrophilicity in the context of folded protein structures

Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc.     

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.