Characterization of a 46 kda insect chitinase from transgenic tobacco

A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.