The T7 promoter and the gene encoding tRNAleu were synthesized chemically and cloned into plasmid pUC19, where the gene was transcribed in vitro with T7 RNA polymerase purified. It was found that spermidine had a nega...The T7 promoter and the gene encoding tRNAleu were synthesized chemically and cloned into plasmid pUC19, where the gene was transcribed in vitro with T7 RNA polymerase purified. It was found that spermidine had a negative effect on T7 transcription. The amount of transcribed tRNA was about 250 times that of the template DNA under optimal reaction conditions. The unmodified tRNAleu1 and tRNAleu2 had similar leucine accepting ability, which however was only one fourth that of the native modified ones. This result indicated that the modified nucleotides of tRNAleu played some though not key roles in its aminoacylation.展开更多
Antarctic cyanobacteria biodiversity was investigated by simultaneous sequencing of the nuclear ribosomal internal transcribed spacer (ITS flanked by partial 16S and 23S), and Chloroplast tRNALeu UAA intron (TrnL), ex...Antarctic cyanobacteria biodiversity was investigated by simultaneous sequencing of the nuclear ribosomal internal transcribed spacer (ITS flanked by partial 16S and 23S), and Chloroplast tRNALeu UAA intron (TrnL), exploring whether such morphotypes constitute distinct species and explaining their current distribution. We identified Nostocales, Chrococcales and Oscillatoriales species, collected in different habitats (soil, algal mats, lake sediments, ice-water) after their growth in cultures. By comparative sequence analyses available in Genbank, our results proved to be mostly in agreement with both TrnL and ITS, in the identification of the strains, particularly for Nostocales. Although ITS demonstrated more usefully than TrnL did in identifying Oscillatoriales and Chroococcales, due to the frequent lack of the intron in these groups, our results lead us to support an independent phylogenetic dataset of ITS and TrnL (the latter based on conserved regions) producing not only concordant clusters but also a secondary structure. Specific assignments of the secondary structure evidenced by different cyanobacteria groups, especially the D1-D1’ region of ITS and the P6b region of TrnL. For the latter region, the sequences analyzed for Nostoc species could be divided into the two classes previously identified, on the basis of different heptanucleotide repeats in P6b which were not展开更多
Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and itsmutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids ...Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and itsmutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal / Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR)展开更多
基金Project supported by the Chinese Academy of Sciences
文摘The T7 promoter and the gene encoding tRNAleu were synthesized chemically and cloned into plasmid pUC19, where the gene was transcribed in vitro with T7 RNA polymerase purified. It was found that spermidine had a negative effect on T7 transcription. The amount of transcribed tRNA was about 250 times that of the template DNA under optimal reaction conditions. The unmodified tRNAleu1 and tRNAleu2 had similar leucine accepting ability, which however was only one fourth that of the native modified ones. This result indicated that the modified nucleotides of tRNAleu played some though not key roles in its aminoacylation.
文摘Antarctic cyanobacteria biodiversity was investigated by simultaneous sequencing of the nuclear ribosomal internal transcribed spacer (ITS flanked by partial 16S and 23S), and Chloroplast tRNALeu UAA intron (TrnL), exploring whether such morphotypes constitute distinct species and explaining their current distribution. We identified Nostocales, Chrococcales and Oscillatoriales species, collected in different habitats (soil, algal mats, lake sediments, ice-water) after their growth in cultures. By comparative sequence analyses available in Genbank, our results proved to be mostly in agreement with both TrnL and ITS, in the identification of the strains, particularly for Nostocales. Although ITS demonstrated more usefully than TrnL did in identifying Oscillatoriales and Chroococcales, due to the frequent lack of the intron in these groups, our results lead us to support an independent phylogenetic dataset of ITS and TrnL (the latter based on conserved regions) producing not only concordant clusters but also a secondary structure. Specific assignments of the secondary structure evidenced by different cyanobacteria groups, especially the D1-D1’ region of ITS and the P6b region of TrnL. For the latter region, the sequences analyzed for Nostoc species could be divided into the two classes previously identified, on the basis of different heptanucleotide repeats in P6b which were not
基金the National Natural Science Foundation of China (Grant No. 39730120) and the Chinese Academy of Sciences.
文摘Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and itsmutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal / Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR)
