After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of...After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.展开更多
AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control...AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.展开更多
Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus...Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein （OPAl）. Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm.展开更多
文摘After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.
文摘AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.
文摘Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein （OPAl）. Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm.
