C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid se...C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.展开更多
目的对候选抑癌基因DOC-1R((Deleted in oral cancer-1 related,CDK2AP2)相关基因DCT1(DOC-1Rterminal1)进行染色体定位分析并观察其细胞内定位,检测该基因在人类组织中的表达。方法通过辐射杂交细胞系对其进行染色体定位分析,同时构建E...目的对候选抑癌基因DOC-1R((Deleted in oral cancer-1 related,CDK2AP2)相关基因DCT1(DOC-1Rterminal1)进行染色体定位分析并观察其细胞内定位,检测该基因在人类组织中的表达。方法通过辐射杂交细胞系对其进行染色体定位分析,同时构建EGFP-DCT1质粒并转染HeLa细胞,荧光显微镜观察DCT1蛋白在细胞内的定位。此外,应用荧光定量PCR的方法检测该基因在16个正常人类组织中的表达。结果DCT1基因定位于5q31,其编码产物在HeLa细胞中定位于核膜,该基因在正常人类的16个组织中均有表达,其中在脾中表达水平较高。结论DCT1可能为细胞周期调控相关基因,在人类组织中普遍表达。展开更多
基金supported by the National Basic Research Priorities Programme(Grant No.001CB108904)the National Natural Science Foundation of China(Grant No.39580001).
文摘C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.
文摘目的对候选抑癌基因DOC-1R((Deleted in oral cancer-1 related,CDK2AP2)相关基因DCT1(DOC-1Rterminal1)进行染色体定位分析并观察其细胞内定位,检测该基因在人类组织中的表达。方法通过辐射杂交细胞系对其进行染色体定位分析,同时构建EGFP-DCT1质粒并转染HeLa细胞,荧光显微镜观察DCT1蛋白在细胞内的定位。此外,应用荧光定量PCR的方法检测该基因在16个正常人类组织中的表达。结果DCT1基因定位于5q31,其编码产物在HeLa细胞中定位于核膜,该基因在正常人类的16个组织中均有表达,其中在脾中表达水平较高。结论DCT1可能为细胞周期调控相关基因,在人类组织中普遍表达。
