Effect of slow pyrolysis conditions on biocarbon yield and properties: Characterization of the volatiles.

Slow pyrolysis of spruce and birch was performed at various heating programs and conditions in a horizontal quartz tube reactor heated by an electric furnace. The effects of feedstock and carbonization conditions on the yield of biocarbon, liquid and gaseous products were studied. The thermal properties, volatile matter (VM) content and the evolution profiles of volatiles from the biocarbons were characterized by thermogravimetry/mass spectrometry. The composition of volatiles was analyzed in detail by pyrolysis-gas chromatography/mass spectrometry. Increased char yield was observed when staged pyrolysis program, low purging flow rate or covered sample holder were applied. Spruce produced more charcoal than birch due to the higher lignin content of softwood. The amount and the evolution profiles of the main gaseous products were similar from spruce and birch biocarbons prepared under the same conditions. The relative amount of aromatic and polyaromatic compounds in VM drastically decreased with increasing carbonization temperature.

The yeast rad18 mutator specifically increases G.C----T.A transversions without reducing correction of G-A or C-T mismatches to G.C pairs.

Inactivation of the Saccharomyces cerevisiae RAD18 gene confers a mutator phenotype. To determine the specificity of this effect, a collection of 212 spontaneous SUP4-o mutants arising in a rad18 strain was characterized by DNA sequencing. Comparison of the resulting mutational spectrum with that for an isogenic wild-type (RAD18) strain revealed that the rad18 mutator specifically enhanced the frequency of single base pair substitutions. Further analysis indicated that an increase in the frequency of G.C----T.A transversions accounted for the elevated SUP4-o mutation frequency. Thus, rad18 is the first eucaryotic mutator found to generate only a particular base pair substitution. The majority of G.C pairs that were not mutated in the rad18 background were at sites where G.C----T.A events can be detected in SUP4-o, suggesting that DNA sequence context influences the rad18 mutator effect. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad18 mutator did not reduce the efficiency of correcting G-A or C-T mismatches to G.C pairs or preferentially correct the mismatches to A.T pairs. We propose that the RAD18 gene product might contribute to the fidelity of DNA replication in S. cerevisiae by involvement in a process that serves to limit the formation of G-A and C-T mismatches at template guanine and cytosine sites during DNA synthesis.

In vitro mutagenesis of the yeast SUP4-o gene to identify all substitutions that can be detected in vivo with the SUP4-o system.

SUP4-o, a suppressor tRNA gene, is the target in a system for characterizing mutational specificity in the yeast Saccharomyces cerevisiae. To date, screening for loss of suppression has detected 172 of the 267 base-pair substitutions possible in the exons and intron of the SUP4-o gene. Although many of the remaining 95 changes might not be detected by this screen, some might occur spontaneously, or be induced, only at very low frequencies. For the purpose of analyzing mutational specificity, it would be valuable to determine which of these substitutions can be detected with the genetic screen employed in this system and which cannot. Thus we used in vitro mutagenesis to generate the 95 substitutions not yet detected in SUP4-o in vivo. Assessment of the phenotypes conferred by these 95 directed mutations revealed that 50 would completely escape detection and only 45 would pass through the first stage of the screen. Of these 45 substitutions, 2 are detectable but have not yet been found among more than 5,000 characterized SUP4-o mutations that arose in vivo. In addition, 4 others should be detected by slightly relaxing the current criteria for selection of SUP4-o mutants. The results indicate that with these modifications the system can detect 174/225 substitutions possible in the SUP4-o exons and 4/42 in the intron.

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase.

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.

Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae.

Disruption of RAD1, a gene controlling excision repair in the yeast Saccharomyces cerevisiae, increased the frequency of spontaneous forward mutation in a plasmid-borne copy of the SUP4-o gene. To characterize this effect in detail, a collection of 249 SUP4-o mutations arising spontaneously in the rad1 strain was analyzed by DNA sequencing. The resulting mutational spectrum was compared with that derived from an examination of 322 spontaneous SUP4-o mutations selected in an isogenic wild-type (RAD1) strain. This comparison revealed that the rad1 mutator phenotype was associated with increases in the frequencies of single-base-pair substitution, single-base-pair deletion, and insertion of the yeast retrotransposon Ty. In the rad1 strain, the relative fractions of these events and their distributions within SUP4-o exhibited features similar to those for spontaneous mutagenesis in the isogenic RAD1 background. The increase in the frequency of Ty insertion argues that Ty transposition can be activated by unrepaired spontaneous DNA damage, which normally would be removed by excision repair. We discuss the possibilities that either translesion synthesis, a reduced fidelity of DNA replication, or a deficiency in mismatch correction might be responsible for the majority of single-base-pair events in the rad1 strain.

Strand interruptions confer strand preference during intracellular correction of a plasmid-borne mismatch in Saccharomyces cerevisiae.

Site-directed mutagenesis was used to construct yeast centromere plasmids in which a strand nick or gap could be placed 5' or 3', on either strand, to a reporter gene (SUP4-o) carrying defined base mismatches. The plasmids were then transformed into yeast cells and the direction and efficiency of mismatch repair were assayed by scoring colouring of the transformant colonies. Strands that were nicked were consistently corrected more often than intact strands, but the effect was very small. However, placement of a small gap at the same positions as the nicks resulted in a marked increase in selection for the gapped strand and an enhanced efficiency of mismatch repair. Both the preference for the gapped strand and correction of the mismatch were offset by deletion of the mismatch repair gene PMS1. Together, the results suggest that strand interruptions can direct intracellular mismatch correction of plasmid-borne base mispairs in yeast.