Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Clonal integration and effects of simulated herbivory in old-field perennials

Summary We compared the growth, phenology and leaf demography of partly defoliated, connected shoots with that of partly defoliated, severed shoots in four old-field perennials (Solidago canadensis, S. altissima, S. gigantea, Aster lanceolatus) with differing genet architectures (rhizome systems), in a common garden and in the field. Our main hypothesis was that defoliation would have fewer negative effects on shoot performance if shoots were connected than if their rhizomes were severed. Since degree of clonal integration is related to differences in genet architecture, our second hypothesis was that the effects of defoliation would be less pronounced in more integrated than in less integrated clones. Removing about 50% of the total leaf area from shoots had different effects depending on plant species, shoot density, and in particular whether rhizome connections between shoots were left intact or severed. In agreement with our prediction, experimentally isolated shoots in the field or in high density clumps in the garden suffered the most from defoliation, while shoots with intact connections or in low density clumps suffered the least. Our second prediction was neither confirmed nor falsified in the present study. Solidago altissima showed overcompensation in response to simulated herbivory in the common garden, i.e. defoliated shoots grew faster and were larger at harvest than their non-defoliated neighbours.     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

recA基因通过同源重组途径参与由整合质粒发动的大肠杆菌染色体复制

我们在前文中报道由整合的F'质粒所发动的大肠杆菌染色体的复制依赖于recA基因。本文报道有关recA、recB、recC以及lexA等在染色体复制中的作用,实验结果说明,recA基因通过同源重组途径而不是通过SOS途径参与复制,而且recA基因和Chi热点无关。实验结果还说明,RecBC酶的依赖于ATP的双链DNA外切核酸酶活性和recA基因的作用无关。