Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol. 相似文献
The plastid gene psbC encodes the CP43 subunit of PSII. Most psbC mRNAs of many organisms possess two possible initiation codons, AUG and GUG, and their coding regions are generally annotated from the upstream AUG. Using a chloroplast in vitro translation system, we show here that translation of the tobacco plastid psbC mRNA initiates from the GUG. This mRNA possesses a long Shine-Dalgarno (SD)-like sequence, GAGGAGGU, nine nucleotides upstream of the GUG. Point mutations in this sequence abolished translation, suggesting that a strong interaction between this extended SD-like sequence and the 3' end of 16S rRNA facilitates translation initiation from the GUG. 相似文献
Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of 439QAAG442 or 443AVYT446 of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors. 相似文献
SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1(-/-) mkk7(-/-) ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1(-/-) ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1(-/-) mkk7(-/-) ES cells. Instead, interleukin-1 beta (IL-1 beta)-induced IL-6 gene expression was greatly suppressed in sek1(-/-) mkk7(-/-) fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages. 相似文献
The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid. 相似文献
One of the rare documented cases of an antagonistic primate–plant interaction is selective foraging by Japanese macaques (Macaca fuscata) on the bark or buds of Japanese mulberry trees (Morus bombycis) in cool-temperate forests. We examined how this selective foraging behavior influences the growth and development of mulberry
trees in a large geographic space with different environmental conditions by selecting study areas in northern Japan. We found
that the foraging caused potentially fatal damage to 5%–10% of the mulberry trees and led to dwarfing of the tree morphology.
However, the stem density of the monitored mulberry trees was the highest in the area with a long history of occupancy by
macaque groups; moreover, the foraging commonly resulted in compensatory plant growth by increasing shoot number. These findings
indicate that the macaque–mulberry relationship is not always antagonistic. Sufficient snow cover could be a key environmental
factor to establish this non-antagonistic interaction by suppressing the negative influence of macaques as a destructive herbivore
and improving their positive influence as a skilful gardener. Finally, we performed decision tree modeling based on the J48
algorithm to investigate geographic variation in mulberry abundance and morphology in response to the distribution of macaques.
We developed an explicit tree model with reasonable predictive performance that not only enables a better understanding of
primate–plant interactions but also provides information regarding the time of occupancy by Japanese macaques in a given area
based on the abundance and morphology of mulberry trees. This result indicated that the observation of preferred tree species
could be an indirect measure that reliably indexes macaque habitat use. 相似文献
Oil-rich algae have promising potential for a next-generation biofuel feedstock. Pseudochoricystis ellipsoidea MBIC 11204, a novel unicellular green algal strain, accumulates a large amount of oil (lipids) in nitrogen-deficient (–N) conditions. Although the oil bodies are easily visualized by lipophilic staining in the cells, little is known about how oil bodies are metabolically synthesized. Clarifying the metabolic profiles in –N conditions is important to understand the physiological mechanisms of lipid accumulations and will be useful to optimize culture conditions efficiently produce industrial oil. Metabolome and lipidome profiles were obtained, respectively, using capillary electrophoresis- and liquid chromatography-mass spectrometry from P. ellipsoidea in both nitrogen-rich (+N; rapid growth) and –N conditions. Relative quantities of more than 300 metabolites were systematically compared between these two conditions. Amino acids in nitrogen assimilation and N-transporting metabolisms were decreased to 1/20 the amount, or less, in –N conditions. In lipid metabolism, the quantities of neutral lipids increased greatly in –N conditions; however, quantities of nearly all the other lipids either decreased or only changed slightly. The morphological changes in +N and –N conditions were also provided by microscopy, and we discuss their relationship to the metabolic changes. This is the first approach to understand the novel algal strain’s metabolism using a combination of wide-scale metabolome analysis and morphological analysis.
HOX cofactors enhance HOX binding affinities and specificities and increase HOX's unique functional activities. The expression
and the regulation of HOX cofactors in human ovaries are unknown. 相似文献