Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62×10^-4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.Abbreviations DOPE dioleoylphosphatidylethanolamine - DOPC dioleoylphosphatidylcholine - Chol cholesterol - OA oleic acid - PEG polyethylene glycol 6000 - NAA -napthaleneacetic acid - BAP 6-benzylaminopurine     

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

Determination of the absolute hand of the helix in the nucleocapsid of haemagglutinating virus (Japan)

The basic hand of the helix of the nucleocapsid of haemagglutinating virus (Japan) was determined to be left-handed from observation of serrated-smooth asymmetry in tilted specimens examined in the electron microscope according to Finch's (1972) technique. Absolute determination of the helical hand was made by comparison with a tilted model of the virus nucleocapsid and by comparing this with the helical sense of the tobacco mosaic virus particle determined by the same method.The left-handed sense of the helix in the nucleocapsid was recognized by examination of cases showing opened-out turns of the helices. Under these conditions the far side of the particle was found to be contrasted predominantly. These conditions are discussed.     

Comparison of isoperoxide patterns in tobacco cell culture and in the intact plant

Analysis by ion-exchange chromatography of the enzymes from cultured tobacco cells and root or leaf tissues of the tobacco plant revealed that the cultured cells contain exclusively cationic peroxidases and the leaf tissues mainly anionic and neutral peroxidases.     

Enzymes of the phenylpropanoid pathway and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus

Pronounced increases in activity of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (CAH) and caffeic acid-O-methyltransferase (OMT)     

Cytokinin-induced shoot formation

Zeatin, (±)-dihydrozeatin and optically active cytokinins (asymmetric carbon α to the exocyclic nitrogen) were tested for their ability to induce development of shoots in tobacco callus. Zeatin and dihydrozeatin were equally active. The levorotatory compounds tested were active in inducing shoot formation but the corresponding dextrorotatory compounds were inactive at all concentrations tested. These findings suggest that the group attached to the N⁶ position of cytokinins binds to a receptor site to bring about organ formation.     

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.     

Protein-RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.