The patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one-dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1-hour heat shock at 44 degrees C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run-on assays of isolated nuclei and cDNA probes to detect hsp70- and hsp88-specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activation of the hsp70 and hsp88 genes. After heat-stressed cells were returned to 37 degrees C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1-hour heat shock at 44 degrees C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37 degrees C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat-sensitive targets from thermal damage. Evidence that the heat-sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half-life). During a 2-hour heat shock, glycerol treatment blocked the heat-induced stabilization of hsp23 RNA and proteins; however, D2O treatment only blocked RNA transcript stabilization, effectively uncoupling the hsp23 protein stabilization pathway from hsp23 RNA stabilization and transcriptional activation of hsp70 and hsp88 genes.

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes L-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone 0-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonialyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid 0-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-0-D-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.

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Synthetic oligonucleotides based on similarity between tobacco 1,3-beta-D-glucanase and barley 1,3-1,4-beta-D-glucanase were used to prime the synthesis and amplification of a 162 bp bean (Phaseolus vulgaris L.) beta-glucanase cDNA by the polymerase chain reaction (PCR). The PCR product was used to isolate a near full-length beta-glucanase cDNA corresponding to an approximately 1400 bp full-length transcript, from a library containing cDNA sequences complementary to mRNA from fungal elicitor-treated bean cells. At the amino acid level, the bean beta-glucanase cDNA was 59% similar to tobacco 1,3-beta-D-glucanase, 46% similar to barley 1,3-beta-D-glucanase and 46% similar to barley 1,3-1,4-beta-D-glucanase. At the nucleotide level, the similarities were 65, 50 and 53% respectively. The beta-glucanase appeared to be encoded by a single gene with similar genomic organization in bean cultivars Canadian Wonder, Imuna and Saxa. On the basis of predicted Mr, isoelectric point, sequence similarity, and comparisons of rate of transcript appearance with induced enzyme activity, it was concluded that the cDNA encodes the basic bean endo-1,3-beta-D-glucanase. Glucanase transcripts were induced, from very low basal levels, with similar kinetics to chitinase transcripts in elicitor-treated bean cell suspension cultures.

*Extracts from-Eswherichia coli cells induced for the adaptive response have been prepared that are capable of repairing 06-methylguanine, 04-methylthymine, and the phosphotriesters produced on the DNA backbone by alkylating-agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The phosphotriesters are repaired by a methyltransferase distinct from the one that demethylates 06-methylguanine. We propose that this increased capacity to repair phosphotriesters accounts for much of the increased resistance to MNNG toxicity seen in cultures induced for the adaptive response.Escherichia coli exposed to sublethal doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) soon become substantially more resistant to both the mutagenic and the cytotoxic effects of MNNG and related alkylating agents (1). This phenomenon is referred to as the adaptive response (2) and is effected by the induction of several proteins. Resistance to the-mutagenic effects of alkylation is mediated by the induction of a protein that rapidly removes one of the major alkylation products, 06-methylguanine (06-MeGua), from the DNA of the alkylated cells (3). This protein is unusual in that it repairs the alkylated guanine base by simply transferring the methyl group from the o6 position of guanine to one of the cysteines of the protein (4, 5). This process leads to inactivation of the enzyme, and any further repair of 06-MeGua requires synthesis of another whole new protein. Such a protein is called a methyltransferase (4).The mechanism-by-which cells develop resistance to the toxicity of MNNG is less well understood. This resistance develops only if the cells have a full complement of DNA polymerase I (6), implicating excision repair in the process in some way. Recently, it has been reported that a DNA glycosylase that is active on 3-methylpurines is induced as part of the adaptive response (7). This glycosylase is the product of the alkA gene (8) and cells mutant in this gene are unable to develop resistance to the toxic effects of methyl methanesulfonate (MMS) (8). These results led to the suggestion that 3-methylguanine might be a potent lethal lesion and its removal, in cells induced for the adaptive response, might be responsible-for the increased survival of these cultures after exposure to alkylating agents (7,8).An alternative explanation for why cells induced for the adaptive response become more resistant to MNNG toxicity is presented in this paper. We have found that cells apparently produce a second methyltransferase in responseto MNNG. This protein does not repair 06-methylguanine but removes methyl groups from the phosphate oxygens of the DNA backbone. We suggest that this dealkylation facilitates a variety of excision-repair reactions the composite effect of which is increased survival. MATERIALS AND METHODSBacterial Strains. The strains used were E. coli B strains F26 (His-Thy-) (2) and two derivatives of this strain-BS21, which is constitutive for the adaptive response (9), and BS21R, which is an Ada-revertant of BS21.Reag...