Dean Ervin Cress

Site-specific mutagenesis of potato spindle tuber viroid cDNA: : Alterations within premelting region 2 that abolish infectivity.

SummaryThe infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C → U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered ‘wild type’ sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [3H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [3H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [3H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca2+ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.

Uptake of Plasmid DNA by Protoplasts from Synchronized Soybean Cell Suspension Cultures

Summary Cell suspension cultures of soybean were synchronized by accumulation of cells at the G 1 /S boundary with 5-fluorodeoxyuridine. Upon removal of fluorodeoxyuridine and addition of thymidine, viable cells resume growth and enter mitosis with increased synchrony. Protoplasts were isolated from cells at varying times following release from inhibition and assayed for uptake of 3 H-pBR322 plasmid DNA. Uptake into DNase-resistant, trichloroacetic acid —insoluble form was 2.5 to 4.5-fold higher in S phase than in G 1 or G 2 phase protoplasts. Endogenous soybean deoxyribonuclease activities did not vary over the cell cycle.

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [3H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.

1063. Independent Modulation of Multiple Genes Using Modified Ecdysone Receptors

The RheoSwitch|[trade]| two-hybrid gene regulation system is the most tightly regulated system available for induction of a transgene, which may be critical for some gene therapy applications. This ecdysone receptor (EcR)-based system uses non-steroidal ligands to induce transgene expression, and they have shown no pleiotropic effects in mammalian cells. Selectivity for ligands of different chemotypes has been created in the ecdysone system by mutating the ligand-binding pocket of the receptor. We have identified and developed multiple ligand-receptor pairs, which respond orthogonally and can be combined to create a multiplexed gene regulation system.