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991.
Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors 总被引:1,自引:0,他引:1
Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes. 相似文献
992.
Navabi ZK Stead KE Pires JC Xiong Z Sharpe AG Parkin IA Rahman MH Good AG 《Genetics》2011,187(3):659-673
Brassica carinata, an allotetraploid with B and C genomes, has a number of traits that would be valuable to introgress into B. napus. Interspecific hybrids were created between B. carinata (BBCC) and B. napus (AACC), using an advanced backcross approach to identify and introgress traits of agronomic interest from the B. carinata genome and to study the genetic changes that occur during the introgression process. We mapped the B and C genomes of B. carinata with SSR markers and observed their introgression into B. napus through a number of backcross generations, focusing on a BC(3) and BC(3)S(1) sibling family. There was close colinearity between the C genomes of B. carinata and B. napus and we provide evidence that B. carinata C chromosomes pair and recombine normally with those of B. napus, suggesting that similar to other Brassica allotetraploids no major chromosomal rearrangements have taken place since the formation of B. carinata. There was no evidence of introgression of the B chromosomes into the A or C chromosomes of B. napus; instead they were inherited as whole linkage groups with the occasional loss of terminal segments and several of the B-genome chromosomes were retained across generations. Several BC(3)S(1) families were analyzed using SSR markers, genomic in situ hybridization (GISH) assays, and chromosome counts to study the inheritance of the B-genome chromosome(s) and their association with morphological traits. Our work provides an analysis of the behavior of chromosomes in an interspecific cross and reinforces the challenges of introgressing novel traits into crop plants. 相似文献
993.
In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtMGAM) of small intestinal maltase glucoamylase (MGAM) are α-glycosidases that catalyze the hydrolysis of α-(1→4) glycosidic linkages in the process of starch digestion, and are considered to be the main therapeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccharides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, voglibose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic α-amylase (HPA) provide us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper, DNJ was the most effective inhibitor against MGAM; the Ki values for the two catalytic domains were 1.41 and 2.04 μM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-03 and 3-03 inhibition constants, Ki, for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02 and 6.08 μM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate specificities and inhibitor tolerance despite their structural relationship. 相似文献
994.
Natural and synthetic steroid hormones excreted into the environment are potentially threatening the population dynamics of all kinds of animals and public health. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. 16S-rRNA analysis showed that bacterial strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the (3)H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5 h and 20 h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in adaptive steroid degradation in Comamonas testosteroni (C. testosteroni), in previous investigations, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established. By this meta-genomic system, two estradiol inducible genes coding 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively, which are involved in steroid degradation, were found in marine strain H5. In the present work, the 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli (E. coli) strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with isopropyl-β-D-thiogalactoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one step using nickel-chelate chromatography. After protein determination, 3-ketosteroid-delta-1-dehydrogenase (0.48 mg/ml) and carboxylesterase (1.28 mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In summary, we have shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA, and purified the corresponding proteins for further research. The exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. The strain might be used for the bioremediation of steroid contaminations in seawater. 相似文献
995.
996.
Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus. 相似文献
997.
Objective
To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs).Methods
Cultured rat VSMCs were exposed to gradient concentrations (10−9-10−5 M) of 17β-estradiol (E2) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10 × 10−6 M) and an estrogen receptor (ER) antagonist ICI 182,780 (10−6 M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively.Results
E2 in its physiological levels from 10−9 M to 10−8 M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10−8 M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E2 increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E2 showed a pure growth-inhibiting effect. The E2 metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E2 over 10−8 M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E2 is due to an increased production of ROS from accumulated E2 metabolites which induces DNA damage, leading to VSMC premature senescence.Conclusion
The complex effect of E2 is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent. 相似文献998.
Production of chitinase from bacteria has distinct advantages over fungi, due to the formation of mycelia of fungi in the
later phase of fermentation. A novel chitinase-producing bacterial strain XJ-01 was isolated from the Yulu fishing field of
Changsha, Hunan province, China, by enrichment and spread-plate technique, sequentially. Physicochemical characterization
and 16S rRNA sequencing revealed that strain XJ-01 belongs to Serratia marcescens. By optimizing the fermentation condition based on L9(34) orthogonal experimental design, a maximal chitinase activity up to 15.36 U/ml was attained by that stain under the condition:
0.5% (NH4)2SO4 as the nitrogen source, 0.75% colloidal chitin as the carbon source, temperature of 32°C, time of 32 h and pH 8.0. 相似文献
999.
Tian YS Xu J Xiong AS Zhao W Fu XY Peng RH Yao QH 《Applied and environmental microbiology》2011,77(23):8409-8414
A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. 相似文献
1000.